Characterization and identification of extracellular polysaccharides-degrading enzymes (epes)-producing marine sediment bacteria

The utilization of polysaccharide-degrading enzymes (EPEs) from bacteria has been increasing, therefore many studies are exploring new producers of EPEs. This study aimed to screen and identify the EPEs-producing marine sediment bacteria collected from Panjang Island, Jepara. A total of 11 bacteria were isolated from the sediment sample. Further, seven strains were selected to conduct further analysis, such as biochemical test and screening of enzyme activity. According to the calculation of enzyme activity index (EAI), it was noted that strain with given codes PP.K.15, PP.K.21, PP.K.6, and PP.K.20 demonstrated potential for carrageenase, alginate-lyase, amylase, and agarase. In addition, molecular identification using 16S rRNA gene sequencing discovered that PP.K.6 was identified as Basillus safensis, PP.K.11 as Sinomicrobium oceani, PP.K. 20 as Salinicola zeshunii, PP.K 15 and PP.K. 21 as Micrococcus luteus, and PP.K 22 as Qipengyuania flava.


Introduction
Enzyme is an important metabolite which produced by an organism as a catalyst to accelerate chemical reaction [1].Extracellular polysaccharide degrading enzymes (EPE) are a class of enzymes capable of catalyzing the degradation of polysaccharides into smaller molecules such as oligosaccharides.Oligosaccharide are known to have better biological activities than their polymers.Hence, some functional food use oligosaccharides in their products [2].These substances could be produced through chemical hydrolysis and biological approach using enzymes [3,4].It is worth mentioning that biological processes using enzymes are relatively safer, more environmentally friendly, and more trusted by consumers.Furthermore, plenty of researches investigated microorganisms as the prospective producer of various important enzymes [5].Notably, marine microbes have captivated many researchers with 1260 (2023) 012056 IOP Publishing doi:10.1088/1755-1315/1260/1/012056 2 their extraordinary potential for enzyme production [6][7][8].In addition, enzymes from marine microbes are noted to be more stabile against temperature, pH, and pressure [7].
Marine bacteria can be isolated from various sources such as living and dead organisms, water, and sediment.Due to the nutrient and biogeochemical cycles that take place in the sediment, it harbors various microbes that helps the cycles [9].Marine microorganisms in sediments will consume organic compounds, by secreting enzymes [10].Sediment accumulates sundry types of organic and inorganic substances in the ocean, include the polysaccharides from marine algae.Compare to fungi, bacteria are noted to have a greater role in marine biogeochemical cycles in the sediment [11].Hence, our study focused on exploration of sediment bacteria that produce EPEs.
Our previous works successfully discovered the EPEs-producing marine microbes that isolated from marine macroalgae [6,7,12].Meanwhile, research of EPEs from marine sediment bacteria is still neglected.Therefore, study of EPS-producing bacteria from marine sediment is very important to be done.This study aimed to screen and identify the EPEs-producing marine sediment bacteria that were collected from Panjang Island, Jepara.

Sediment sampling
The sediment samples were collected around seaweed beds from Panjang Island, Jepara region, Central Java, Indonesia (Figure 1).The samples were put in zip lock plastic and brought to the laboratory for bacterial isolation.

Bacterial isolation and purification
Bacteria isolation was done according to Sarker et al. [13] with modification on the medium.This study utilized a modified medium called MT with compositions such as, Peptone 0.2 %, Yeast Extract 0.1%, Meat Extract 0.1%, Calcium Carbonate (CaCO3) 0.1 %, Glucose 0.5%, Starch 0.5%, NaCl 0.2%, Agar 2.0%, and Nystatin.The sediment was spread evenly onto MT medium to strain the bacteria.The plates were incubated for two to four weeks at 26°C.The strains were maintained on MT agar.

morphological and biochemical characterization
The strains were characterized based on their margin, shape, elevation, and colony colour.Further, the gram staining, motility test, sugar fermentation, citrate test, and catalase test were performed to characterize its biochemical characteristics [14][15][16].

Enzyme Activity Index
EPEs assay was done by observing the presence of a clear zone around the bacterial colonies on special media containing certain substrates such as agar, alginate, carrageenan, and starch.The media were composed according to our previous works [6,8].Each plate was incubated at 26 ℃ for 7 days.After incubation, a Lugol solution was poured into each plate for 30 second.The presence of clear zone indicated a positive result.The enzyme activity index was obtained by calculating the ratio between the clear zone and the diameter of the strain colony using the EAI formula as below:

EAI=
Clear zone diameter-Colony diameter Colony diameter

Molecular Identification
DNA extraction was done using Zymo Quick-DNA Miniprep Kit DNA Extraction protocol.The DNA amplification was performed with PCR mix consisting of 1 μL of primer 27F (5'AGAGTTTGATCMTGGCTCAG-3), 1 μL of primer 1492R (5'GGTTACCTTGTTACGACTT-3'), 1 μl of DNA template, 12.5 μl of GoTaq® Green Master Mix Promega, and 9.5 μl of ddH2O.Then, the DNA was amplified using BIO-RAD T100 Thermal cycler [8].The PCR products were sent to The 1 st Base (Malaysia).The bacterial species was identified using BLAST homology by comparing the DNA sequence to the gene bank in NCBI.A phylogenic tree was reconstructed using Mega X.

Result and discussion
Plenty of microorganism have been discovered from Panjang Island ecosystem, from prokaryotic like bacteria to eukaryotic microorganisms such as fungi [17][18][19] .Our previous study has succeeded in obtaining various species of association bacteria from this location such as Salinicoccus roseus, Sphingobium yanoikuyae, Streptomyces lateritius, Labrenzia marina, and Halomonas meridiana [20,21].However, research on marine sediment bacteria has not been carried out.Marine sediment were formed from various contents due to the oceanographic and biogeochemical phenomenon [22].In addition, the substances in the sediment are utilized by bacteria for their metabolism [23].In this study, eleven strain were obtained from the isolation and have been characterized by morphological and biochemical identification.The result of the bacterial characterization is presented in Table 1 and Table 2. Morphological, cells morphology, and biochemical characteristics data were used to classification the group of the bacteria [21].According to gram staining, eight strain were identified as gram-positive bacteria and three other strain were identified as gram-negative bacteria.The identification of the gram stain method is determined based on the color that appears under the microscope.The colored of the bacteria is caused by their composition of the peptidoglycan and lipid layer of the cell wall [16].Gramnegative bacteria have lipopolysaccharides in their cell walls that prevent the peptidoglycan from binding to the crystal violet solution.This lipopolysaccharide will dissolve when rinsed with decolorized solution, so that the peptidoglycan will be free to bind with safranin then it gives a red or pink color to the cells.On the other hand, gram-positive bacteria have a thick peptidoglycan layer which give a purple color as a result of bonding with crystal violet.The peptidoglycan will be not dissolved in the decolorized solution therefore the gram-positive bacteria will retain the purple color [24].
Citrate test was done in Simmon Citrate Agar (SCA medium) to observe the ability of bacteria to use citrate as a carbon source.The positive control were indicated by the color change in the media after 24 h incubation [25].Citrate is utilized by bacteria to obtain carbon and energy source.Bacteria which consume the citrate will produce the citrate-permease and citrate lyase to covert citrate into pyruvate [26].The blue color that appeared in the citrate test was caused by the alkaline condition after the citrate transformation [27].
Sugar fermentation test was conducted to identify the ability of the bacteria to ferment glucose, lactose and sucrose.Four strain such as PP.K.6, PP.K.7, PP.K.10, and PP.K.17 were showed the ability to fermented glucose which characterized by a color change on the bottom/butt of the medium from red into yellow color [28].The strain that only ferment glucose will produce acidic compound on the butt of media in limited quantity so that they cannot change the color of the surface of the media.On the other side, isolates that ferment sucrose, lactose, or both will produce acidic compound that can change the surface color.The color change is caused by phenol red indicator which detect acidic conditions such as PP.K.8 [29].
Each strain had the ability to produce catalase, this is evidenced by the appearance of bubble when the colony was dripped with hydrogen peroxide(H2O2) [30].Catalase is an enzyme that degrades H2O2 into water (H2O) an oxygen (O2).The bubbles that appeared in the catalase test were oxygen gas (O2) from the degradation product.In addition, some of pathogenic bacteria produce catalase as a form of defense [31].Polysaccharides degrading enzyme (EPEs) assay was done in semi agar medium with the addition of appropriate composition such as amylase, alginate-lyase, agarase, and carrageenase.The result of enzyme activity screening is shown in Table 3.The presence of EPEs production is indicated by the appearance of a clear zone around the bacterial colony after flooded by Lugol.Lugol will enter into the helix structure of polysaccharides and give a dark color.Polysaccharides that are degraded due to enzyme activity cannot bind Lugol and form a clear zone [32].Apart from PP.K.8 and PP.K.12, other strain showed the ability to produce EPEs.This is accordance with previous studies which proved that EPEs are capable of being produced by marine sediment bacteria [2].Enzyme production by bacteria can be caused by various factor such as DNA expression, the source composition, and habitat condition [33][34][35].
Six strain were selected to proceed to the next stage, such as calculation of enzyme activity index (EAI) and molecular identification.Enzyme activity index (EAI) was calculated based on the ratio between the clear zone and strain's colony diameter.The results of EAI are shown in the Figure 2.   The selected strain were identified using 16S rRNA gene sequencing.PP.K.6 was identified as Bacillus safensis subsp.safensis with 99.45% similarity.This strain showed the enzyme activity in all polysaccharide-enriched media with the best alginate-lyase activity with EAI score 3.55.Previous studies have proven that Bacillus spp.have an ability to many of enzyme include amylase, but the research of Bacillus safensis subsp.safensis potential still lack to find [36][37][38].In other hand, PP.K.15 and PP.K. 21 were identified as Micrococcus luteus with 99.93% and 98.94 % similarity.This two strain showed different enzyme activities, such as the carrageenase abilty of PP.K.15 was the best among other isolate with an index of 2.87 while PP.K.21 had the best alginat lyase with an index of 2.42 (Figure 3).M. luteus has been proven to have the ability to produce several enzymes such as protease and peptidase [39].However, research on its ability to produce EPEs has not been carried out.Based on the EAI calculation, PP.K.20 has the best agarase enzyme ability with an index value of 1.77.This strain was identified as Salinicola zeshunii with 96.19% similarity.The ability of this strain was obtained in this study is in accordance with similarity studies that have been carried out [8].This present study showed than PP.K.11 and PP.K.22 strains were capable of producing more than one EPEs.Based on molecular identification, PP.K.11 was indicated as Sinomicrobium oceani with 100% similarity and PP.K.22 was indicated as Qipengyuania flava with 99.42% similarity.Interestingly, there was no any report about these bacteria for their EPEs production capability.

Figure 4 .
Figure 4. Phylogenic tree of the selected strain.

Table 1 .
Macroscopic characteristics of marine sediment bacteria from Panjang Island.

Table 2 .
Cells and biochemical characteristics of marine sediment bacteria from Panjang Island.

Table 3 .
The result of EPEs screening.

Tabel 4 .
Molecular identification of the selected EPEs-producing marine sediment bacteria.