Production of Protoplasts from Yeast Cells (Saccharomyces cerevisae) Isolated from Palm Wine (Elaeuis guinensis) Using Snail Gut Enzyme from African Giant Snail (Achatina achatina)

Abstract

Aims: The aim of this study was to investigate the use of locally sourced snail gut enzyme from African giant snail (Achatina achatina) for yeast protoplasts production. Place and Duration of Study: Fresh undiluted palm wine was commercially acquired from Umuoke, Obowo in Imo State while African giant snails (A. achatina) were bought from Umuahia main market, Abia State, Nigeria. The study was carried out from June to september, 2013. Methodology: The snails were starved for 7 days so as to concentrate their gut juice, carefully aspirated using sterile needles and syringes after unshelling the snails. All the extracts from twelve snails were pooled together and centrifuged at 1,500rpm for 20 minutes to remove larger particulates; the supernatant was collected dissolved in 0.1M acetate buffer (pH 5.5). Yeast cells (Saccharomyces cerevisae) were grown in potato dextrose broth and harvested at late exponential phase at 37ºC. 1g of the cell slurry were suspended into 10 ml of 0.1M phosphate buffer (7.2) containing 0.1% Tween 80(osmotic stabilizer). Crude snail gut enzyme extract in 0.1M acetate buffer (pH 5.5) was added to yeast suspension and incubated at 30°C for 120 mins with gentle shaking. The resulting protoplasts were centrifuged at (1000rpm) and suspended in 0.7Mol. NaCl solution as supplement osmotic medium. The effect of lytic incubation time, concentration and osmotic stabilizers were studied. The viability of the cells were assessed by regeneration method. Results: A wet mount of the harvested yeast cells after staining with lactophenol cotton blue indicate presence of cell walls. However, after incubation of mixture of these cells with snail gut juice at 30°C for 120 mins. The effect of lytic incubation time for the protoplast production shown on Fig. 1, revealed optimal protoplast formation of 2.7 x 107/mL after 80mins of incubation. The enzyme concentration effect on protoplast production shown in Fig. 2 revealed that 100%v/v of gut juice from A. achatina was able to produce 2.0 x 107/mL yeast protoplast. The best supporting osmotic stabilizer during cell wall regeneration was observed with 0.5M sucrose. Conclusion: This experiment therefore suggests a rapid, inexpensive and efficient procedure for yeast protoplasts production