World Journal of Microbiology and Biotechnology, 8, 313--315

Cellulases of Vibrio agar-liquefaciens isolated from sea mud V.K. Abhaykumar* and H.C. Dube Vibrio agar-liquefaciens produced the component enzymes of a cellulase complex (exoglucanase, endoglucanase and ~/-glucosidase). Whilst complete cellulase activity occurred in three media, each containing carboxymethylcellulose as the sole carbon source, different activities of the individual enzymes resulted. The enzymes showed a temporal sequence in their activity and differed in pH and temperature optima. NaCl enhanced the enzyme activities to varying degrees at different substrate concentrations. Key words: Cellulase, endoglucanase, exoglucanase, glucosidase, Vibrio agar-liquefaciens

Marine vibrios can use and degrade a wide variety of organic compounds, including cellulose (Mouzouras et al. 1986). Thus, they play a vital role in the carbon cycle of the seas. As part of the mixed microflora in coastal environments, they colonize wood piling and form a 'primary film' that attracts fouling and boring macroorganisms (Austin et aI. 1979). A study of cellulolytic degradation by vibrio is essential to combat biodegradation of materials and to maintain the ecological health of the marine environment (Colwell 1979). In this study we have examined Vibrio agar-liquefaciens isolated from the tropical muddy coast of Bhavnagar (Gujarat) for cellulase production in different media at different pH values and the effect of various physical and chemical factors on its activity.

Materials and Methods The Organism Vibrio agar-liquefaciens was isolated from deteriorating wooden pilings exposed to seawater on the muddy coast at the port of Bhavnagar. It was grown on Kadota medium (Kadota 1959) and purified and identified using standard methods.

Enzyme Production The culture was grown on three media: (i) Kadota medium contained KzHPO4 1 g; NaNO 3 0.5 g; MgSO4.7H20 0.5 g; FeSO4.7HzO

V.K. Abhaykumar and H.C Oube are with the Department of Life Sciences, 8havnagar University, Bhavnagar, Gujarat, India. * Corresponding author.

0.01 g; carboxyrnethylcetlulose (medium viscosity, degree of substitution 0.05, Hercules Powder Co., USA) I0 g; aged seawater I000 ml; (ii) ZoBell medium contained peptone 5 g; yeast extract I g; FePO4 0.01 g; carboxymethylcelulose I0 g and aged seawater 1000 m[; (iii) Man&Is and 5ternberg medium contained (NH4)2SO4 1.4; KH2PO4 2 g; urea 0.3 g; CaCl2 0.3 g; MgSO4.7H20 0.3 g; FeSO4.7HzO 0.005 g; Tween 80 0.033%; carboxymethylcellulose I0 g and aged seawater I000 mt. Media, in 50-ml volumes, were inoculated with I ml of the bacterial suspension (106 cells/ml) incubated at room temperature (32 q-2~ and culture filtrates were examined for cellulase production from day 2 to day 12. Residual cellulose was removed by centrifugation at 10,000 x g for 15 rain and the supematants were passed through a 0.45/am filter to produce a cell-free enzyme solution.

Enzyme Assay The component enzymes of the, cellulase complex, exoglucanase (Avicelase) (EC 3.1.2.91), endoglucanase (CMCase) (EC 3.2.1.4), fl-glucosidase (EC 3.2.1.21) as well as complete cellulase (filter paper activity), were assayed in triplicate by the method of Mandels et al. (I976) using, respectively, Avicel, carboxymethylcellulose, salicin and filter paper. The enzyme activities are expressed in international units (IU) (/amol glucose released per rain per ml).

Results and Discussion All three cultivation media supported production of cellulase enzymes (Table 1). No single medium supported the highest production of all the component enzymes or the complete cellulase. Exoglucanase was highest in Mandels and Sternberg medium while the other two enzymes were maximally produced in the Kadota medium.

9 1992 Rapid Communications of Oxford Ltd

World Journal o[ Microbiology and Biotechnology, Vol 8, I992

3 I3

V.K. Abhaykumar and H.C. Dube Table 1. Activities of cellulase enzymes (In IU/ml) In the culture filtrates of Vibrio agar-liquefaclens grown on three media. Medium

Days

Exoglucanase

Endoglucanase

~-Glucosidase

Complete cellulese

Kadota

2 4 6 8 10 12

0.11 0.13 0.15 0.15 0.14 0.13

0.06 0.07 0.08 0.09 0.09 0.07

0.03 0.04 0.05 0.06 0.07 0.06

0.00 0.01 0.01 0.01 0.02 0.01

Mandels and Sternberg

2 4 6 8 10 12

0.11 0.16 0.19 0.20 0.18 0.17

0.06 0.06 0.07 0.09 0.08 0.07

0.03 0.04 0.05 0.06 0.05 0.05

0.00 0.01 0.01 0.01 0.01 0.00

ZoBell

2 4 6 8 10 12

0.11 0.12 0.15 0.17 0.18 0.15

0.04 0.06 0.07 0.07 0.09 0.08

0.02 0.03 0.04 0.05 0.06 0.05

0.01 0.02 0.02 0.01 0.02 0.02

0.22 0.20 0.18 0.16 0.14

|

E

3

0.12 0.10 0.08 0.06 0.04 0.02 0.0C pH

>I----

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Cellulases ofVibrio agar-liquefaciens isolated from sea mud.

Vibrio agar-liquefaciens produced the component enzymes of a cellulase complex (exoglucanase, endoglucanase and β-glucosidase). Whilst complete cellul...
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