Background and Objectives: Enzymes are selective proteins that catalyze all metabolic reactions found in living organisms. Millions of enzymes are produced by prokaryotes and eukaryotes. Marine sponges have developed a enormous quantity of diverse microorganisms such as bacteria, fungi, viruses, protozoa, and single-celled algae and the nature of the sponge-microbe interaction is diverse. Marine sponge's symbiotic bacteria can comprise as much as 40% of sponge tissue volume. At least 32 bacterial phyla and candidate phyla were described from marine sponges by both cultivation-dependent and cultivation-independent techniques. Symbiotic microorganisms in sponges can be sources of various natural products, because metabolites previously ascribed to sponges have recently been demonstrated to be biosynthesized by symbionts. If symbiotic microorganisms from which some natural products are derived can be cultured, the microorganism could be used in a mass production of the bioactive comopounds. The goal of this research is isolation of Amylolytic bacteria from marine sponges and optimization of bacterial growth and amylase production in most amylolytic isolate. Symbiot bacteria were isolated using serial dilution method and differential analyses done for isolated bacteria. Most effective isolate was selected by using qualitative starch hydrolysis method.
Methods: Morphological and biochemical characteristics were studied using Bergey’s manual of systematic bacteriology. Differential analyses were performed by catalase and Gram test TSI, H₂S, VP, MR tests. Preliminary screening and quantification of amylase activities were analysed in selected isolates by starch agar plate and dinitrosalicylic acid (DNS) method respectively. In order to optimize the enzyme production, the fermentation process was carried out under M9 medium using various sodium chloride concentrations (2–14%). To evaluate the effect of pH on the production of Amylase, the best amylase producer isolate was cultured at M9 medium with different pH rang 4-8. After incubation time it was subjected to amylase activity assay. Isolate SS1 was grown in M1 medium at 28°C for 7 days. DNA extraction was done using Cinnapure DNA Kit for isolation of DNA from gram positive bacteria. PCR amplification of 16S rRNA gene was performed, the specific actinomycete primers, F27 and R1492 were used to amplify 16S rDNA. The resultant 16S rRNA gene sequences were aligned manually with corresponding almost complete sequences. The 16S rRNA gene sequence search was performed using the BLAST program available from the National Centre for Biotechnology Information (NCBI) and the isolate was identified to generic level Using the CLUSTAL-X Multiple Sequence Alignment Program (Strasburg, France), The Phylogenetic tree was constructed by Neighbor-Joining method using MEGA 6.06. The reliability of the phylogenetic tree was tested by bootstrap analysis using 1,000 replicates.
Findings: Biochemical analyses showed that all tests were catalase and Gram positive none of them produced H2S and TSI, MR, VP tests were negative. According to 16S rRNA sequences result the most effective isolate was belong to Bacillus genus and Optimum growth condition was found at 12% NaCl concentration, 35˚C temperature and pH 12. Maximum amylase activity in optimum condition was 109 Unit (one Unit is amount of enzyme that release one microgram of glucose in a minute).
Conclusion: The results showed that enzyme production is related to growth of bacteria conditions and the amylase gene should amplify from selected isolate and clone in suitable host for more studies.