Ocky Karna Radjasa

Highlights of marine invertebrate-derived biosynthetic products: Their biomedical potential and possible production by microbial associants

Coral reefs are among the most productive marine ecosystems and are the source of a large group of structurally unique biosynthetic products. Annual reviews of marine natural products continue to illustrate that the most prolific source of bioactive compounds consist of coral reef invertebrates-sponges, ascidians, mollusks, and bryozoans. This account examines recent milestone developments pertaining to compounds from invertebrates designated as therapeutic leads for biomedical discovery. The focus is on the secondary metabolites, their inspirational structural scaffolds and the possible role of micro-organism associants in their biosynthesis. Also important are the increasing concerns regarding the collection of reef invertebrates for the discovery process. The case examples considered here will be useful to insure that future research to unearth bioactive invertebrate-derived compounds will be carried out in a sustainable and environmentally conscious fashion. Our account begins with some observations pertaining to the natural history of these organisms. Many still believe that a serious obstacle to the ultimate development of a marine natural product isolated from coral reef invertebrates is the problem of compound supply. Recent achievements through total synthesis can now be drawn on to forcefully cast this myth aside. The tools of semisynthesis of complex natural products or insights from SAR efforts to simplify an active pharmacophore are at hand and demand discussion. Equally exciting is the prospect that invertebrate-associated micro-organisms may represent the next frontier to accelerate the development of high priority therapeutic candidates. Currently in the United States there are two FDA approved marine-derived therapeutic drugs and two others that are often cited as being marine-inspired. This record will be examined first followed by an analysis of a dozen of our favorite examples of coral reef invertebrate natural products having therapeutic potential. The record of using complex scaffolds of marine invertebrate products as the starting point for development will be reviewed by considering eight case examples. The potential promise of developing invertebrate-derived micro-organisms as the starting point for further exploration of therapeutically relevant structures is considered. Also significant is the circumstance that there are some 14 sponge-derived compounds that are available to facilitate fundamental biological investigations.

Raw starch–degrading α‐amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

The aims were to isolate a raw starch–degrading α‐amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli.

Construction of individual, fused, and co-expressed proteins of endoglucanase and β-glucosidase for hydrolyzing sugarcane bagasse.

At least a combination of endoglucanase (EglII) and β-glucosidase (BglZ) is required for hydrolyzing crystalline cellulose. To understand the catalytic efficiency of combination enzymes for converting biomass to sugars, EglII and BglZ were constructed in the form of individual, fused as well as co-expression proteins, and their activities for hydrolyzing sugarcane bagasse were evaluated. The genes, eglII isolated from Bacillus amyloliquefaciens PSM3.1 earlier and bglZ from B. amyloliquefaciens ABBD, were expressed extracellularly in Bacillus megaterium MS941. EglII exhibited both exoglucanase and endoglucanase activities, and BglZ belonging to the glycoside hydrolase 1 family (GH 1) showed β-glucosidase activity. A combination of EglII and BglZ showed activity on substrates Avicel, CMC and sugarcane bagasse. Specifically for hydrolyzing sugarcane bagasse, fused protein (fus-EglII+BglZ), co-expression protein (coex-BglZ+EglII), and mixed-individual protein (mix-EglII+BglZ) produced cellobiose as the main product, along with a small amount of glucose. The amount of reducing sugars released from the hydrolyzing bleached sugarcane bagasse (BSB) using fus-EglII+BglZ and mix-EglII+BglZ was 2.7- and 4.2-fold higher, respectively, than steamed sugarcane bagasse (SSB), indicating the synergetic enzymes worked better on treated sugarcane bagasse. Compared with fus-EglII+BglZ and mix-EglII+BglZ, coex-BglZ+EglII released more mol reducing sugars from SSB, indicating the enzymes were potential for biomass conversion. Additionally, coex-BglZ+EglII acted on BSB 2.5-fold faster than fus-EglII+BglZ. Thus, coex-bglZ+eglII expression system was the best choice to produce enzymes for hydrolyzing sugarcane baggase.

ANTIBACTERIAL PROPERTY OF A CORAL-ASSOCIATED BACTERIUM PSEUDOALTEROMONAS LUTEOVIOLACEA AGAINST SHRIMP PATHOGENIC VIBRIO HARVEYI (IN VITRO STUDY)

A coral-associated bacterium was successfully screened for secondary metabolites production based on PCR amplification of the nonribosomal peptide synthetase gene and was identified as closely related to Pseudoalteromonas luteoviolacea based on its 16S rDNA. The bacterium was found to inhibit the growth of shrimp pathogenic bacterium tested, Vibrio harveyi. To characterize the inhibiting metabolite, a 279 bp long DNA fragment was obtained and the deduced amino acid sequence showed conserved signature regions for peptide synthetases and revealed a high similarity to NosD (40% identity), a multifunctional peptide synthetase from Nostoc sp. GSV224, and NdaB (44% identity), a peptide synthetase module of Nodularia spumigena

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13.

Isolation, Identification And Screening Antibacterial Activity from Marine Sponge-Associated Fungi Against Multidrug-Resistant (MDR) Escherichia coli

Irrational used of antibiotic in several decades ago causing resistant in bacteria and decreasing the cure rate of infectious diseases. Multidrug-resistant (MDR) Escherichia coli is known to cause various of infectious diseases such as urinary tract infection, nosocomial bloodstream infection, meningitis, bacteraemia, and gastrointestinal disease. Marine sponge-associated fungi have potential as source of new compound to combat MDR E. coli. The aims of this research were to isolate marine sponge-assosiated fungi, to screen potential fungi against MDR E. coli, to identify the potential fungi and its host sponge. There were 29 marine sponge-associated fungi successfully isolated from 9 sponges. Among 29 sponge-associated fungi screened, there were 7 isolates showed antibacterial activity against MDR E. coli. The best inhibition zone produced by MPS 14.1/MT 02 and MPS 14.3/MT 04 from sponge PP.SP.16.14. According to fungi identification result fungus MPS 14.1/MT 02 was identified as Trichoderma asperellum while MPS 14.3/MT 04 was identified as Trichoderma reesei. Sponge identification leaded the PP.SP.16.14 as Cinachyrella sp.

Genetic diversity of the causative agent of ice-ice disease of the seaweed Kappaphycus alvarezii from Karimunjawa island, Indonesia

An essential step in investigating the bacterial role in the occurrence of diseases in Kappaphycus alvarezii is the characterization of bacteria associated with this seaweed. A molecular characterization was conducted on the genetic diversity of the causative agents of ice-ice disease associated with K. alvarezii widely known as the main source of kappa carrageenan. K. alvrezii infected with ice-ice were collected from the Karimunjawa island, North Java Sea, Indonesia. Using Zobell 2216E marine agar medium, nine bacterial species were isolated from the infected seaweed. The molecular characterizations revealed that the isolated bacteria causing ice-ice disease were closely related to the genera of Alteromonas, Bacillus, Pseudomonas, Pseudoalteromonas, Glaciecola, Aurantimonas, and Rhodococcus. In order to identify the symptoms causative organisms, the isolated bacterial species were cultured and were evaluated for their pathogenity. Out of 9 species, only 3 isolates were able to cause the ice-ice symptoms and consisted of Alteromonas macleodii, Pseudoalteromonas issachenkonii and Aurantimonas coralicida. A. macleodii showed the highest pathogenity.

Screening of Antimicrobial Activity of Sponges Extract from Pasir Putih, East Java (Indonesia)

The emergence of new infectious diseases, the resurgence of several infections that appeared to have been controlled, and the increase in bacterial resistance have created the necessity for studies directed towards the development of new antimicrobials; considering the failure to acquire new molecules with antimicrobial properties from marine sponges. The objective of this study was to evaluate screening of antimicrobial activity of seven sponges extract from Pasir Putih, East Java against some Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and Gram negative (Escherichia coli and Klebsiella pneumoniae) as well as drug-resistant bacteria (S. aureus and Pseudomonas aeruginosae) using the agar diffusion method and phytochemical screening of the extract. The findings show that the extract, Xestospongia testudinaria has a stronger antibacterial activity against bacterial pathogens S. aureus, E.coli, K. pneumoniae, Salmonella typhi and bacteria resistant P. aeruginosa MDR and S. aureus MRSA compared to other sponge extract. In conclusion, the showed X. testudinaria ethanol extract was more active than other sponge extracts.

Symbiotic Fungus of Marine Sponge Axinella sp. Producing Antibacterial Agent

The emerging of multidrug resistance pathogenic bacteria cause the treatment of the diseaseshave become ineffective. There for, invention of a new drug with novel mode of action is an essential for curing the disease caused by an MDR pathogen. Marine fungi is prolific source of bioactive compound that has not been well explored. This study aim to obtain the marine sponges-associated fungus that producing anti-MDR bacteria substaces. We collected the sponge from Riung water, NTT, Indonesia. The fungus was isolated with affixed method, followed with purification with streak method. The overlay and disk diffusion agar methods were applied for bioactivity test for the isolate and the extract, respectively. Molecular analysis was employed for identification of the isolate. The sponge was identified based on morphological and spicular analysis. The ovelay test showed that the isolate KN15-3 active against the MDR Staphylococcus aureus and Eschericia coli. The extract of the cultured KN15-3 was also inhibited the S. aureus and E. coli with inhibition zone 2.95 mm and 4.13 mm, respectively. Based on the molecular analysis, the fungus was identified as Aspergillus sydowii. While the sponge was identified as Axinella sp.

Exploration of Fungal Association From Hard Coral Against Pathogen MDR Staphylococcus haemolyticus

Staphylococcus haemolyticus are opportunistic bacteria and as the second leading cause of nosocomial infections. It is a disease causing septicemia, peritonitis, otitis, and urinary tract infections and infections of the eye. It also a phenotype resistant to multiple antibiotics commercial. There is now an urgency to find an alternative antibiotics to combat this bacteria. It has been widely reported that many bioactive marine natural products from marine invertebrate have striking similarities to metabolites of their associated microorganisms including fungi. Hard coral associated microorganisms are among of the most interesting and promising marine natural product sources, which produce with various biological activities. The proposed work focused on the discovery of bioactive compounds and also estimated the phylogenetic diversity from fungal association of hard coral against pathogen MDR Staphylococcus haemolyticus. A total of 32 fungal association, FHP 7 which were isolated from Favia sp. capable of inhibiting the growth MDR. Molecular identification based on 18S rRNA gene sequences revealed that the active fungal association belonged 100% to the members from one of the genera Trichoderma longibrachiatum. Accession Number LC185084.1.