Production of Extracellular Anti-leukaemic AntiEnzyme L-Asparaginase from Marine LActinomycetes by Solid-state and SolidSubmerged Fermentation: Purification and Characterisation

Kelompok 4 : Mei Ria S 8151 Rista Susanti 8157 Novyani Nurhayati 8161 Etika Dyah P 8177 Ika Nugrahaning P 8180 Fikri Budi M 8185

INTRODUCTION
Marine microbes represent a potential source for commercially important bioactive compounds. y One of them is Actinomycetes. y Actinomycetes have gained special importance as the most potent source of antibiotics and other bioactive secondary metabolites.
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L-asparaginase (Lasparaginase aminohydrolase, E.C.3.5.1.1) discovered in 1922 as a medicinal agent for the treatment of malignant tumors. y L-asparaginase was found at Guinea Pig Serum (Clementi), Escherichia coli (Masburn & Wrinston), Erwinia aroidae and Serratia marcescens.
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L-asparaginase converts L-aspargine to Laspartic acid. y Several types of tumour cells require Lasparagine for protein synthesis.
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MATERIALS
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Marine sediment samples were y collected from different sites in y Tamilnadu and Kerala, India at a y depth of 10 cm in May 2007 Polythene bags Spatula Starch, y Casein, y Yeast extract Peptone, L-asparagine, y Soybean meal Beef extract

Acrylamide, Bisacrylamide Bovine serum albumin and commassie brilliant blue G-250 were purchased from Hi-media (Mumbai, India). Sephadex G-100 was obtained from Sigma- Aldrich, Germany Richloroacetic acid was purchased from E. Merck Ltd, Mumbai, India. All other chemicals were of analytical grade and used without further modification.

METHOD
Enrichment and isolation of marine microorganism Characterisation of marine International Streptomyces morphological identification staining and hanging drop characterisation soil isolate using standard Project (ISP) procedure, by simple staining, Gram method and biochemical

Screening of soil isolates for L-asparginase production by rapid-plate assay pink zone around colonies (sample no. S3, S4, K8) Crude enzyme production by solid state fermentation (SSF) and submerged isolation (SF) optimalisation to get crude enzyme

Protein determination the absorbance was measured at 660 nm using double beam UV-visible spectrofotometer and the protein content determined. Determination of L-Asparginase activity assay enzyme method. The rate of hydrolysis of L-asparginase was determined by measuring the ammonia released using Nesslers reaction. Purification of L-Asparginase from marine actinomycetes A total of 30 fractions were collected at the flow rate 5 ml/30 min. Fractions showing high activity were pooled and uses for further studies.

Characterisation of partially purified LAsparginase using effect of pH, temperature and metal ions. Determination of Km value and Vmax by Lineweaver-Burk plot

RESULTS
From the 20 marine sediment samples tested, only 10 sediments showed growth in enrichment media.

SCREENING OF L-ASPARAGINASE LPOSITIVE CULTURES

Of the isolated Streptomyces spp. Screened for L-asparaginase activity, only S3 and S4 from Tamilnadu and K8 from Kerala showed positive results in rapid plate assay method.

PRODUCTION OF L-ASPARAGINASE
All three isolates examined synthesized asparaginase with yields ranging from 24.61 to 49.23 U/ml. y Isolate S3 was selected for further studies because of its high productivity (49.23 U/ml). y The concentration of protein obtained from isolated organism S3 was found 65 g/ml.
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PURIFICATION OF L-ASPARAGINASE L-

CHARACTERIZATION OF PARTIALLY PURIFIED L-ASPARGINASE L-

Enzyme exhibited maximum activity between pH 7.0 and 8.0 with 80% activity at physiological pH. The optimum temperature was 50 C. The isolated enzyme was activated by Mg+, but inhibited by Cu+, Zn+ and EDTA.

DETERMINATION of Km and Vmax

The Km and Vmax of partially purified enzyme were approximately 24 M and 51 IU/ml.

DISCUSSION
Although L-asparaginase from bacteria has been extensively characterized, a similar attention has not been paid to actinomycetes. To date, antibiotics are the major bioactive compounds obtained from actinomycetes. y Soil isolates S3 and S4 from Tamilnadu and soil isolate K8 from Kerala possess Lasparaginase activity. y This indicates that the formation of pink zone is due only to L-asparaginase production.
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Among the three media used, the solid-state media gave the highest enzyme activity for all the 3 isolates presence of glucose metabolic products. Studies suggest that in the case of asparaginase biosynthesis, the depressive effect of carbohydrates may be a function of their ability to lower the pH value of the fermentation media. A comparison of submerged and solid-state fermentation shows a significant difference in enzyme activity.

This suggests that there may be increased accumulation of intermediate metabolites between substrate and product formation in submerged fermentation. In the final purification step, the enzyme showed a specific activity of 662.61 IU/mg, which is approximately 2-fold purity. Optimum pH was found to be 7.5, which is close to blood pH,

At 50 C, the enzyme showed its optimum activity. The test results of the sensitivity of Lasparaginase to heavy metal ions indicate that the activity of the enzyme may depend on the presence of sulfhydryl functional groups. The affinity of L-asparaginase to its substrate is related to its degree of effectiveness against tumors. The substrate affinity of L-asparaginase as measured by Km value was found to be 2.4 x 10-5 M for L-asparagine. This compares favorably with Km values reported for antineoplastic Lasparaginases from other sources such as E. coli (1.25 x 10-5 M), Proteus vulgaris (2.6 x 10-5 M) and Erwinia aroideae NRRL Q-138 (3.0 x 10-5 M).

The linearity of the Lineweaver-Burk double reciprocal plot was suggests that the isolated L-asparaginase followed the Michaelis-Menten kinetics.

CONCLUSION
Marine sediment samples from Tamilnadu and Kerala in India are potential sources of bioactive actinomycetes. y Hyperproductive marine actinomycetes with higher L-asparaginase activity was successfully isolated using a low-cost substrate -soyabean meal. y L-asparaginase appears to be a promising agent and requires further investigation of its potential anti-leukaemic activity.
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REFERENCES
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Basha, N. S., R Rekha., M Komala., and S Ruby. 2009. Production of Extracellular Anti-leukaemic Enzyme LAsparaginase from Marine Actinomycetes by Solid-state and Submerged Fermentation: Purification and Characterisation. Tropical Journal of Pharmaceutical Research. 4 :353-360

Doni 8074: Apakah ada kaitan antara luas zona pink yang terbentuk dengan tingginya nilai pengukuran dengan spektrofotometri? Substratnya apa (untuk spektrofotometri)? Konversi nilai amonia dg L-asparginae? Fitri 8127: Bagaimana penggunaan enzim ini untuk pengobatan? Annas : metode fermentasinya?

7891 Yuda : SF Setengah cair atau seperti apa? 7979 Dini : Aktifitas opt. 50oC, bagaimana caranya agar dapat digunakan sebagai anti leukimia? 8086 Dewi : Amonia merupakan hasil dari aktifitas enzim tersebut atau merupakan hasil dari assay pada metode tersebut?