Maggy Thenawidjaja Suhartono

Purification and Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than 60°C. Fibrinolytic activity was increased by 5 mM MgCl2 and 5 mM CaCl2 but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the α and β chains of fibrinogen but was unable to degrade the γ chain.

Fermentation RS3 derived from sago and rice starch with Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629

Resistant starch type 3 (RS3) is retrograded starch which is not digested by human starch degrading enzyme, and will thus undergo bacterial degradation in the colon. The main fermentation products are the Short Chain Fatty Acid (SCFA): acetate, propionate and butyrate. SCFA has significant benefit impact on the metabolism of the host. The objectives of this research were to study the SCFA profile produced by colonic butyrate producing bacteria grown in medium containing RS3. RS3 was made from sago or rice starch treated with amylase, pullulanase and the combination of amylase and pullulanase. Fermentation study was performed by using Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629, which has been identified as capable of degradation of starch residue and also regarded as beneficial bacteria. Experimental result revealed that enzyme hydrolysis of retrograded sago or rice starch was beneficial to RS formation. RS3 derived from sago contained higher RS (31-38%) than those derived from rice starch (21-26%). This study indicated that C. butyricum BCC B2571 produced acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1, when the medium was supplemented with RSSA at concentration 1%. In the medium containing similar substrate, E. rectale DSM 17629 produced acetate, propionate and butyrate at molar ratio of 1.7 : 1 : 1.2. High levels of acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1.1 was also produced by E. rectale DSM 17629 in medium supplemented with RSSP at concentration 1%. The results showed that both bacteria responded differently to the RS3 supplementation. Such result provided insight into the possibility of designing RS3 as prebiotic with featured regarding SCFA released in the human colon with potential health implication.

Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO4, 1.4% K2HPO4, 0.02% CaCl2·2H2O, 0.002% FeSO4·7H2O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F2 fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70°C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90°C, the t1/2 values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90°C, the enzyme retained its activity up to 8 h in the presence of 1mM MnCl2. The enzymes activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The Km app values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the Vmax values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F2 chitosanase at 24 h incubation (70°C) were pentasaccharide (GlcN)5 and hexasaccharide (GlcN)6. The prelimiaary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium. Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.

Stability and growth characteristics of GFPuv-labeled Cronobacter sakazakii isolated from foods

Stability of ultraviolet green fluorescent protein (GFPuv)-labeled Cronobacter sakazakii and C. muytjensii isolated from foods and the effects of the plasmid GFPuv (pGFPuv) on growth were analyzed. PCR analysis and microscopic observation showed that C. sakazakii and C. muytjensii isolates took up the plasmid into cells and expressed the GFPuv gene. All Cronobacter transformants maintained this plasmid after frozen storage and 2 consecutive subcultures. The C. sakazakii FWHd16 transformant was the most stable, while the C. muytjensii FWHd11 transformant was the least stable. All transformants showed nearly identical growth curves during lag, log and stationary phases, compared to wild type parental isolates. The maximum bacterial growth rates (μmax) of the transformants and parents were similar, indicating that the presence of pGFPuv in transformants did not affect cell growth. Stable GFPuv-labeled Cronobacter has potential for use in tracking bacterial behavior during food handling and drying.

Thrombus Degradation by Fibrinolytic Enzyme of Stenotrophomonas sp. Originated from Indonesian Soybean-Based Fermented Food on Wistar Rats.

Objective. To evaluate thrombus degrading effect of a fibrinolytic enzyme from food origin Stenotrophomonas sp. of Indonesia. Methods. Prior to animal study, the enzyme safety was tested using cell culture. The effect on expression of tissue plasminogen activator was also analysed in the cell culture. For in vivo studies, 25 Wistar rats were used: normal control, negative control, treatment groups with crude and semipurified enzyme given orally at 25 mg/kg, and positive control group which received Lumbrokinase at 25 mg/kg. Blood clot in the tail was induced by kappa carrageenan injection at 1 mg/kg BW. Results. Experiment with cell culture confirmed the enzyme safety at the concentration used and increased expression of tPA. Decreasing of thrombus was observed in the positive group down to 70.35 ± 23.11% of the negative control animals (100%). The thrombus observed in the crude enzyme treatment was down to 56.99 ± 15.95% and 71.5 ± 15.7% for semipurified enzyme. Scanning electron microscopy showed clearly that bood clots were found in the animals injected with kappa carrageenan; however, in the treatment and positive groups, the clot was much reduced. Conclusions. Oral treatment of enzyme from Stenotrophomonas sp. of Indonesian fermented food was capable of degrading thrombus induced in Wistar rats.

Biochemical characteristics of chitinase enzyme from Bacillus sp. of Kamojang Crater, Indonesia.

Chitinase and chitindeacetylase are enzymes capable of degrading chitin into chitooligomers and chitosan. The chitinases characterized and purified in this study were extracted from the acidophillic Bacillus sp. isolated from Kamojang Crater West Java Indonesia. When grown in liquid media containing colloidal chitin, the optimum chitinase activity of the acidophilic isolate was reached after 4-5 days of incubation. The optimum temperature and pH of the chitinase and chitin deacetylase were found at 37 degrees C and pH 5. When incubated at pH 5, the activity of chitin deacetylase was increased; after 3 h, the activity was 1.5 times of the control. The enzyme was stable at pH 4, after 2 h incubation, the activity was still 80% of the control. The chitinase and chitin deacetylase activities were not influenced by Mg(++) nor Ca(++), Ni(++) and Cu(++) inhibited the chitinase activity, while chitin deacetylase activity was not affected by Cu(++) addition. When 1 mM of EDTA was added, the enzyme activity was reduced 40 to 50%.

Characterization of transposon-generated protease mutant of Xanthomonas campestris pathovar glycine 8ra. Enzyme activity, cloning, and mapping of flanking DNA.

Protease negative mutant ofXanthomonas campestris pathovar glycine 8ra (prt- mutant) was constructed by mutagenesis employing artificial transposon Omegon-Km. Transposon delivery was conducted through diparental conjugation usingX. campestris pathovar glycine 8ra as recipient andEscherichia coli S17-1 carrying pJFF 3500 plasmid as the donor. The frequency of transconjugation was found 1.9 x 10-7 per recipient. Enzyme analysis indicated the presence of mutant with lower protease activity than that of the wild-type.Genetic analysis employing pulsed-field gel electrophoresis (PFGE) of the genomic DNA digested withAseI orSpeI restriction endonuclease could significantly differentiateX. campestris pathovar glycine 8raprt- from the wild-type parent. The 9.85 kb pLR Ω 6 plasmid was constructed from the genomic DNA of theprt- mutant after being digested withKpnI restriction endonuclease and ligated with T4 DNA ligase.

Protease of Stenotrophomonas sp. from Indonesian fermented food: gene cloning and analysis

Screening of proteolytic and fibrinolytic bacteria from Indonesian soy bean based fermented food Oncom revealed several potential isolates. Based on 16s rDNA gene analysis, one particular isolate with the highest proteolytic and fibrinolytic activity was identified as Stenotrophomonas sp. The protease gene was amplified to generate a 1749 bp Polymerase Chain Reaction product and BLAST analysis, revealed 90% homology with gene encoding protease enzyme from Stenotrophomonas maltophilia. The putative amino acid sequence indicated a serine protease enzyme with typical amino acid aspartate, histidine and serine in the catalytic triad. The gene was translated into a pre-pro-protein consisted of cleavage site on its N terminal and Pre-Peptidase Cterminal domain. Cloning of the protease gene in pET22b with Escherichia coli BL21 DE3 as the host showed that the gene was expressed as insoluble protein fraction. This is the first report for analysis of protease gene from food origin Stenotrophomonas sp.

Keratinolytic Enzymes for Cleaning Edible Bird’s Nest

Edible bird’s nest (EBN) as a kind of functional foodhashigheconomic value depending on the qualitysuch as color and hygiene. The purpose of this research was to find optimum condition for application of keratinolytic enzymesBacillus sp. MTS in cleaning EBN.Activating agents for both enzymes were cationdivalents, EDTA, reducing agents, organic solutions, and antibacterial agents.Additives compound that able to increase keratinase activity were used to make cleaning solution and its tested on EBN and human hair. Alcoholic solutions (25% ethanol, 25% methanol, 25% glycerol), and some divalent metallic ions(Ca2+, Mg2+, Mn2+, Zn2+)were able to increase keratinasewhile disulfide reductase was solely activated by 0.05 mM EDTA. The activity of both enzymes wasinhibited by NaCland Na-azide.The activity of keratinase of Bacillus sp. MTS in cleaning solution formulated in this research was 2-3 fold as much as control (crude extract) in human hair substrates. Gliserol and cations divalent increasing 2-3 fold keratinase activity in cleaning solution. The solution was successfully apllied to cleaning EBN with weight loss 2.3-2.5% approximately.

Identification of Pathogenecity of Avian Influenza Virus Subtype H5N1 from Waterfowls Base on Amino Acid Sequence of Cleavage Site Hemagglutinin Protein

Identification of pathotype of Avian Influenza Virus (AIV) subtype H5N1 isolates is very important. This research aimed to identify the pathotype of AIV subtype H5N1 isolated from household waterfowls in West Java based on molecular markers of amino acid sequences of the Hemagglutinin (HA) cleavage site. Fragments of HA genes of 21 isolates were amplified using RT-PCR with a primer pair that flanking the cleavage site region, and sequenced with dideoxy-termination method with ABI automatic sequencer (Applied Biosystems). Multiple alignment of nucleotide and their deduced amino acid sequence were analyzed using ClustalW from MEGA 3.1 program. The result shows that all H5N1 isolates (21 isolates) possess polybasic cleavage sites with 2 patterns of amino acid sequence, i.e QRERRRKKR (20 isolates) and QRESRRKKR (1 isolate). This finding indicates that all of the viruses isolated in this research were of highly pathogenic avian influenza (HPAI) strains. Keywords: cleavage site, waterfowls, HPAI