Estimation of Chlorophyll
On 7th, 15th, 30th, 45th, and 60th day, 100 mg of leaves of the plants was weighed and homogenized with 2 ml of 80-% acetone. Then the volume was made up to 10 ml using distilled water. The contents were centrifuged at 5000 rpm for 15 minutes and the supernatants were taken for the absorbance analysis using UV ‘ Visible Spectrophotometer (Hitachi U 3210), at 663 nm and 645 nm. The chlorophyll contents were calculated by using the formula:
Total chlorophyll (mg/g tissue)
20.2 (A645) + 8.02 (A663) X V/1000 X W
A = Absorbance at specific wave lengths (645 nm and 663 nm)
B = Final volume of chlorophyll extract in 80% acetone and
W = Fresh weight of tisssue extracted.
Chloropyll ‘ a
12.7 (A663) ‘ 2.69 (A645) X V/1000 X W
Chlorophyll – b
22.9 (A645) ‘ 4.68 (A663) X V/1000 X W
Strain properties and identification
The agarolytic strain is a Gram-positive rod shaped bacterium, motile; it is also catalase, oxidase and lysine decarboxylase positive, and oxidase and Phosphatase negative. The preliminary identification results showed that the isolated strain was in accordance with Bacillus, according to Bergey’s Manual of Systematic Bacteriology. The results are shown in Table 1.
The 16S rDNA sequence of the agaro degrading strain RV3 was compared to available sequences in public databases. Fig. 1 shows an un rooted tree of the Bacillus species. The isolated strain and B. subtilis strain ZJ-06 formed a robust clade. Based on data results, we assigned of our strain as B. subtilis st. RV3. The 16S rDNA sequence of Bacillus subtilis strain RV3 was submitted to the GenBank database under the accession number GQ413935.
Purification of agarase
The enzyme has high binding affinity to DEAE-cellulose when loaded at low salt concentrations. The enzyme was released gradually from the DEAE-cellulose column by washing with 1.5 M NaCl. Further purification of the agarase was accomplished by gel filtration using Sephadex. The specific enzyme activity was increased from 0.22 units/mg protein to 5.3 units/mg protein after purification (Table S1).
Agarase from B. subtilis st. RV3 has a molecular weight of 65 kDa, as analyzed by a comparison with the of protein standard markers (Fig. 2).
Physiochemical properties of the enzyme
Agarase enzyme purified in the present investigation was found to be active in a broad range of temperature ranging from 10~60??C. However, optimum temperature was found to be 30??C (Fig. 3a). The pH report of agarase of B. subtilis strain RV3 had a maximum activity at pH 5 and 7.5 (Fig. 3b). Agarase activity was assayed in the presence and absence of metal ions. It was observed that none of the cations exhibited activation in the enzyme activity (Table.2).
The agarase stored at 20??C exhibited a decline in agarase activity by 20% within three days, which was totally lost within 6 days whereas the one stored at room temperature (30??C) exhibited a 48% decline in enzyme activity within 3 days which …
...(download the rest of the essay above)