ANTIBACTERIAL POTENCY OF CHITOSAN OLIGOMER PRODUCED BY Bacillus licheniformis MB-2 CHITOSANASE

Ekowati Chasanahi), Meidina2), and Maggy T. Suhartono3) 'l ) Marine Biotechnology Lab, Center for Marine and Fisheries Product Processing and Biotechnology Research-Petamburan, Jakarta 2) Food Science Program, Graduate School, BogorAgricultural University-Bogor, Indonesia 3) Department of Food Science Technology, Faculty of Agricultural Technology, BogorAgricultural University-Bogor, Indonesia Received september T;1331;.-3""?,1i"f ii-"#:t rorm March 25-2008;


INTRODUCTION
Chitosan is a natural polymer containing N-acetyl-D-glucosamine and D-glucosam ine resid ues. Havin g biodegradable and biocompatible properties, this deacetylated form of chitin polymer has been used widely at various industries such as food, agricultural, pharmaceutical, and waste water treatments. To improve its application, i.e enhancing the water solubility properties, the polymer is usually modified by substituting the functional groups with chemical substances or partially hydrolyzed chemically or enzymatically. Application of enzyme for partially degrading chitosan was more preferable because of the mild and safe process as well as specific reaction, resulting in high quality oligomer products. Specific size oligomer was reported to maintain various prominent biological activities such as antimicrobial properties.
Partially degraded chitosan or chitosan oligomer was reported to have antibacterial properties against some pathogenic bacteria compared to the native one (Kendra & Hadwiger, 1984). The antibacterial mechanism has been proposed as loosing of barrier function of bacterial cell wall caused bv chitosan binding to the outer membrane of bacteria, and Chung et al. (2004) reported that there was a positive relationship between antibacterial activity of chitosan and surface characteristics of bacterial cell wall.
Another possible mechanism through inactivation of important enzymes such as protease has never been reported yet.
Bacillus licheniformis MB-2 has been isolated from hot spring water of Manado and be able to produce thermostable chitosanase. In pure form, the enzyme hydrolyzes specifically chitosan, producing pentamer and hexamer of chitooligosaccharide (Chasanah, 2004). Preliminary study on antibacterial properties of oligomers from the crude enzyme using 4 concentration (0.005 tp 0.170 U per mg chitosan) showed that oligomer produced by applying 0.1 U per mg chitosan and reaction time of 1 to 3 hours was able to reduce the 6 pathogenic bacteria tested (Meidina et a\.,2004). Further study on the sensitivity and mechanism of antibacterialthrough their potency as anti protease was assesed in this study using P aeruginosa protease. Six pathogenic bacteria frequently associated with food borne infection and intoxication was used.

Purification
Cell free supernatant containing 30% satura+;d ammonium sulphate (75 ml) was applied -.rnto hydrophobic Butyl Sepharose 4 fastflow matrix, which had been previously equilibrated with 30% saturated ammonium sulphate to 0.05 m phosphate bufer (pH6). Aftersample application, the column was washed with the same buffer, and the eluate was obtained by washing with linear gradient of 1S% saturated ammonium sulphate phosphate buffer(0.05 m, pH 6) and 0.05 m phosphate buffer (without ammonium sulphate) with elution rate of 2 ml per h. Each fraction (3 ml)was collected, and the protein and chitosanase activity were measu red.

Ghitosanase Assay
Chitosanase assay was conducted according to Yoon ef a/. (33), with modifications. The reaction mixture consisting of 100 ilof 1% colloidalchitosan, 100 il 0.05 m phosphate buffer (pH 6) and 100 it of the enzyme solution was incubated at optimum temperature for 30 min. The reaction was stopped by incubating the mixture at -10"C for 15 min. The amount of reducing sugar in the mixture was determined by a modified method of Schales (27). Anamount of 200 il of the solution \ivas further mixed with 1 ml schales reagent and 800 il aquadest, and are further heated in boiling water for 15 minutes, centrifuged for 10 min at 3,000 g and the absorbance was read at 6=420 nm. A YZ blank was prepared using aquadest. One unit of chitosanase activity was defined as the amount of the enzyme which produces '1 imol of reducing sugar (glucosamine) per minute.

Protein Determination
Protein contentwas determined based on Bradford method using bovine serum albumin as the protein standard at 0.2 to 1.2m9 protein per ml. The reaction mixturecontained 100ilof sample, l mlof aquadest and 1 ml Bradford reagent. Aftervortexing the mixture, the absorbance was read at 595 nm. A blank was prepared by substituting sample solution with 100 il of aquadest.

Chitosan Oligomer Preparation
Oligomer chitosan was made by reacting the enzyme (0.1 unit per mg chitosan), both crude and pure, to 1 of 85% deacetylated chitosan for t hour (Meidina et al., 2004). Oligomer was harvested by boiling the mixture to inactivate the enzyme followed by sentrifugation (10,000 rpm, 10 minutes). The oligomer (supernatant) was sterilized a|121"C, 15 minutes before applied for antibacterial test.

Viability Study of Pathogenic Bacteria
Bacterialviability was analyzed by contact method (Carson & Riley, 1995). Bacterial culture amounting to 104CFU per mlwere incubated along with chitosan oligomer, and incubated in medium broth at 37'C. The number of bacteria at 0, 1, 3, 11, and 24 hours were plated and counted as colony forming unit per ml The chitosan oligomer concentration used was 1 and 1.5 MIC value (Meidina et al., 2004) ldentification of Chitosan Oligomer Chitosan oligomerformed was identified using dual lambda 440 mode HPLC, with 60% acetonitrile solvent in water as the moving phase. Detection was based on retention time. The flow rate speed used was 1 ml per min. The oligosaccharides standard (monomer to hexamer of glucosamine) was used at 1% (wlw).

Chitosan oligomer as anti protease
Protease enzyme was produced by inoculating 1 ose of Pseudomonas aeruginosa into LB and incubated 'for 24 h in 37"C shaker water bath. Extracellular protease was harvested by centrifuging the broth at 10,000 g for 15 min. Protease was assayed using 2% casein according to Bergmeyer ef a/ (1983).

Enzyme and Oligomer Production
Bacillus licheniformis MB-2 chitosanase was harvested at the seventh days of fermentation using 15% starter, at 55'C 120 rpm shaker bath. Crude enzyme was obtained by concentrating the cellfree supernatan with 80% saturated ammonium sulphate.
Purification was done by hydrophobic interaction colomn chromatography as previously mention, and the relatively pure enzyme (Figure 1, Fraction 2) was collected, Oligomer chitosan was made by reacting the enzyme, both crude, and pure, to 1 of 85% deacetylated chitosan. Optimal concentration of the crude enzyme effectively retarded the 6 pathogenic bacteria tested was 0.1 lU per mg chitosan with reaction time of 1 to 3 h (Meidina et a/., 2004). The diameter of a clear zone as antibacterial indication was about 10 to 19 mm for the 6 pathogenic bacteria-This concentration was used in this experiment with reaction time of t h. For pure enzyme, the same concentration was used. In this experiment, oligomer possesing antibacterial properties was identified based on HPLC analysis. lt was found that oligomer mixture were in the from of 2 to 6 unit oligomer.

Antibacterial StudY
Previous study by Meidina resulted that MIC value of oligomer produced by the crude chitosanase, was lower (321 to 562 ppm) compared to the polymer chitosan (7,000 to 1 0,000 ppm), but it was much higher compared to the commercial antibiotics (5 to 100 ppm).
The MIC value of Satmonella typhymurium was 321 ppm, while the MIC ol Pseudomonas aeruginosa, Staphytococcus aureus, and Escherichia coli were 402 ppm, and the MIC of Listeria monocytogenes and Baciltuscereus were 482 and 562 ppm, respectively.
In this study, antibacterial potency of the oligomer was assesed by viability study. The concentration of oligomer used was at the MIC and 1.5 MlC, and the nuriberof bacteria survived was counted during certain while Esche richia coti and Sa/rnonella typhimurium were significantly reduced by 11 and24 hours contact' On the other hand, gram positive pathogenic bacteria tested were reduced not as much as those gram negative bacteria. The results was in agree with lnd.Fish  previous research results (Chung et al., 2004', Helander et al., 2001. lt was reported that there was a positive relationship between antibacterial activity of chitosan and surface characteristics of bacteria cell wall. Gram negative bacteria which has more negative charged and more hydrophilic were more sensitive to oligomer chitosan compared to the gram positive bacteria. Chitosan oligomer produced by pure chitosanase did not give different inhibition results as the crude one.
,f*n It was hypothized that protease secreted by pathogenic bacteria was known as toxin, and some other were hydrolytic enzymes capable of degrading 94 extracellular matrix compounds of host polymer, for their carbon and energy sources.
In this experiment, chitosan oligomer produced by Bacillus licheniformis MB-2 was assessed for antiprotease. Results showed that chitosan oligomer at their MIC were able to retard 64% of Pseudomonas 4 Contact time (hours) Antibacterial Potency of Chitosan ..... by Baciltus Licheniformis MB-2 Chitosanase (Chasanah, E., et al.) aeruginosa protease having activity of 0.09 U per ml, while 25% retardation was obtained when using chitosan oligomer at 0.5 MlC. From the result, it can be deduced that the ability of chitosan oligomer, produced by Bacillus licheniformis MB-2 chitosanase, to inhibit pathogenic bacteria might through antiprotease mechan ism. coNcLUstoNs 1. Chitosan oligomers produced by Bacillus ticheniformis MB-2 chitosanase has capability of reducing the 6 pathogenic bacterial tested by 2lo 5 log CFU per ml during 24 h contact.
2. Gram negative bacteria were more sensitive to the chitosan oligomer than the gram positive one.
3. The oligomers, at MIC value, was able to inhibit Pseudomonas aeruginosa protease by 64oh, suggesting that the ability to inactivate bacterial protease might be one of the antimicrobial mechanisms performed by chitosan oligomer.