Screening of β-galactosidase producing fungi from marine samples

Fungi are used in many industrial processes, such as the production of enzymes, vitamins, polysaccharides, polyhydric alcohols, pigments, lipids, and glycol-lipids. Some of these products are produced commercially while others are potentially valuable in biotechnology. Fungal secondary metabolites are extremely important to our health and nutrition and have tremendous economic impact. The screening and isolation procedures resulted, five potentially interesting fungal isolates (MF S2-02, MF S3-09, MF S4-11, MF S5-08, and MF S6-09). All were able to produce more then 50 U/gm DCW of intracellular activity and 10 U /ml extracellular activity. The results indicate that the isolate MF S4-11 is promising strain with the yield of 117 U/gm DCW. A good number of media that were reported in the literature were screened to select optimal production media and medium number ‘IV’ was found to be the best for the production of â-galactosidase (128 U/ gm DCW).


INTRODUCTION
The ocean remains as an unexploited source for many drugs and pharmacologically active substances.K Kathiresan 1 et al (2008) isolated marine lactic acid bacteria.Pseudomonas BAL-31 is a marine Pseudomonas first isolated from samples of seawater taken from the Pacific Ocean by Espejo and Canelo 2 (1977).
No reports are available concerning the presence of β-galactosidase producing fungi from marine environment.Marine environment may yield strains which may produce novel metabolites or potent producers of known metabolites.
β-galactosidase, is the enzyme widely used especially in dairy & food technologies.This enzyme provides two benefits, which make its use attractive in industries: 1) Preparation of lactose-oligosaccharides that are interesting from the technological as well as health perspective.Low activity of β-galactosidase causes digestive insufficiency, called lactose intolerance.Decreased activity of β-galactosidase is rather frequent especially with seniors.Lactose-free milk is manufactured for those patients (Suarez 3  There are no reports on the production of βgalactosidase from marine fungi.In view of this, it is proposed to isolate and screen marine fungi for the detection of β-galactosidase producing fungi.

Samples
Seven marine samples were collected from Bay of Bengal.Out of seven samples, two were marine sediment samples, two were marine water samples and remaining three were marine sand samples.The sediments were collected with the core sampler near Vishakhapatnam (one at R.K beach and other at Gangavaram beach).These were brought to the lab and processed immediately.

Isolation of fungi
About 1 gm of each sample was transferred in to 50 ml sterile marine water (250 ml EM flask), agitated on rotary shaker for one hour to get uniform suspension and serially diluted.One ml of each appropriate dilution was plated (6 inch diameter plates) on to 50 ml molten PDA and YEME media, and allowed to set and incubated at 30 0 C for 7 days.Both PDA and YEME dehydrated media were procured from Himedia.
Pure and selected colonies were transferred on to PDA slants and incubated at 30 0 C for 7 days.The cultures were preserved at 4 0 C and sub-cultured monthly.
About 72 fungal colonies in total were isolated from the above samples.The isolates were pooled together and the cultures which appear identical to the naked eye with respective colour of the aerial mycelium, reverse mycelium colour, soluble pigments and surface texture were eliminated.54 different fungal colonies were selected from total 72 colonies.

Screening of the marine fungal isolates for âgalactosidase activity
The selected 54 isolates were tested for their ability to grow on PDA medium containing lactose as a sole source of carbon.Plates were incubated at 28 0 C for 5 days.The isolates were inoculated, incubated at 28 0 C for 5 days and 22 isolates were selected based on the extent of growth.The selected 22 isolates were initially screened for the β-galactosidase producing capacity by using ONPG dicks.Each isolate was plated on to PDA medium and incubated at 28 0 C for 5 days.Colonies from each plate were picked up and transferred to a test tube of (0.1 ml) sterile saline solution.The sterile ONPG disk (Sigma) was transferred aseptically to the above saline solution.They were incubated at 28 0 C for 24 hr.The colonies producing β-Galactosidase turned the solution to yellow colour by releasing Ortho-nitro phenol (ONP).The intensity of the colour depends up on ability of the isolate to produce β-galactosidase.Out of 22 fungal isolates, 9 isolates were not able to release ONP and they were considered to be non-producers and 13 isolates were found to be producers of the enzyme.Among these 13, five potent isolates were selected on the basis of the intensity of yellow colour.

Micro-organisms
All the five promising isolates were grown on a PDA slants with lactose as a carbon source for 7 days at 28 0 C.

Submerged fermentation Spore suspension Preparation
From the full grown slant the spores were suspended in 10 ml of distilled water containing 0.01% Triton X-100.
At the end of the incubation the cell mass was collected by centrifuging the medium in a refrigerated centrifuge (4 0 C) at 3000 rpm for 20 minutes.The cell mass was washed twice with sterile distilled water, suspended in 10 ml of sterile distilled water and used as inoculum.

Production of β β β β β-galactosidase
The inoculum (10% level) was transferred aseptically to 100 ml production medium contained in 500 ml EM flask.The composition of the production medium is same as above except dextrose.The flasks were incubated on a rotary shaker (150 rpm) at 28 0 C for 5 days.
The samples (5 ml) were withdrawn for every 24 hr and centrifuged at 1500 rpm for 15 minutes.The clear supernatant was separated and the cell mass was used for the detection of intracellular enzyme activity.Cell free supernatant (CFS) was used for the detection of extracellular enzyme activity.About 0.5 gm of cell mass was used to determine the intracellular enzyme activity.The enzyme assay was carried out as described below.For intracellular activity 0.5 gm of cells were treated with 0.5 ml of toluene at 37 0 C for 5 minutes. 2 ml of ONPG (4mg/ml) prepared in citrate buffer (pH 4.6) was added and incubated at 40 0 C for 15 minutes.The reaction was stopped by the addition of 2.5 ml 10% (w/v) Na 2 CO 3 .The release of O-nitro phenol from ONPG was measured at 420 nm with the help of spectrophotometer (UV-VTS Spectrometer 117 from Systronics).Similarly extracellular enzyme activity was measured using 1ml aliquots of cell free supernatant (CFS).

Fig. 1: The typical fermentation course of the isolate MF S4-11 is depicted
The results of intracellular and extracellular activities of the selected isolates are presented in the Table 1.3.
Among the five isolates MF S4-11 exhibited the highest activity.It had both the intercellular and extracellular activity.The intercellular activity is three times more than the extracellular activity.

Determination of MDW
For the determination of mycelial dry weight, 10 ml aliquots of the sample was with drawn from the medium, Centrifuged the cells for 15 minutes.Decant the supernatant liquid and washed the cells with sterile distilled water two times to remove the medium components.Carefully transferred the cells in to pre weighed aluminum foil.These cells were dried to a constant weight at 80 0 C. Data were averaged with three trails.

pH profile of the isolate MF S4-11
pH was measured for all the 24 hr samples up to the 5 days.No considerable change was observed.

Enzyme activity
The isolate MF S4-11 showed maximum enzyme activity at 110 hr.

RESULTS AND DISCUSSION
The marine strains may have better potential than their terrestrial counterparts.This is due to essential nutrients provided by marine biotopes for nurturing the micro organisms, and to extreme environmental niches.The marine fungi and their by-products may have potential values in food processing, fermentation, pharmaceutical and biopolymer industries.Marine samples were collected from Bay of Bengal near Vishakhapatnam and the samples were screened for marine fungi which resulted, 72 isolates.The fungal isolates were tested for the presence of β-galactosidase activity.Among 72 isolates, 22 isolates exhibited positive response for β-galactosidase activity (both intracellular and extracellular enzyme), of which 5 isolates were found to be potent enzyme producers.
As all the isolates are producing more quantity of intracellular enzyme, subsequent studies were devoted to extract endo-enzyme.These isolates were further tested for their β-galactosidase production capabilities by submerged fermentation.The strain MF S4-11 was selected fro the production of β-galactosidase based on the hydrolysis of ONPG.The fermentation studies indicated that the maximum enzyme production at 110 hours of fermentation (Fig 1 .1).

CONCLUSION
The results of this study showed that ocean is a great source of microorganisms, with the possibility of screening of microbes for all kinds of biotechnological uses.The screening and isolation procedures for β-galactosidase from marine source resulted, five potential fungal strains with both intra and extra cellular activity.More than 72 fungal strains were screened for the presence of β-galactosidase.Out of them the isolate MF S2-11 was chosen for further isolation and purification of the enzyme βgalactosidase.