Yeast culture and PCL films, foam plastics degradation assay

The Pseudozyma strain was grown on the yeast malt agar (YMA) (glucose 1.0 % w/v, peptone 0.5 % w/v, yeast extract 0.3 % w/v, malt extract 0.3 % w/v, agar 2.0 % w/v), and the plates were incubated for 2 days at 25 °C. A single colony of yeast species was inoculated into 5 ml of YM broth and cultured at 30 °C. After 24 h, 1 ml of culture broth was inoculated into 1,000-ml flask contain 50 ml YM broth and incubated at 30 °C with shaking at 200 rpm over night. Sterilized dry PCL film of known weight was added to each culture in addition to positive control (YM broth culture of each Pseudozyma species without PCL film) and negative control (YM broth with PCL film only). Flasks were incubated at 30 °C with shaking at 200 rpm for 15 days. Three replicas from each flask have been done considering the different time intervals (3, 5, 8, 12 and 15 days) to study the degradation behaviour. Under the same conditions, foam plastics were incubated in yeast culture but for longer time (30 days). The PCL films and foams were taken out carefully in different intervals time, washed thoroughly by double distilled water to remove any media components or yeast cells if present on the their surface and were completely vacuum dried at 30 °C over night. The percentage of weight loss was recorded.

Culture turbidity

Growth rate of yeast culture incubated with and without PCL films and foams was measured and assayed according to the culture turbidity (OD_600nm).

pH values

Effect of variable pH values on the growth rate, protein concentrations and enzyme activity have been studied.

Preparation of polycaprolactone (PCL) film and market’s foam plastic

Poly (ε-caprolactone) (PCL) with molecular weight 70,000–100,000 Da was purchased from Sigma-Aldrich Chemical Co. PCL film was prepared in small glass petri dishes (3 cm) by dissolving polymer in acetone and left for air drying. The prepared PCL films and the 1 cm quadrate cutting foams (polystyrene with molecular weight 100,000–400,000 Da) were weighed and sterilized in 80 % ethanol for 2 min, well dried and then kept under UV lamp for 30 min and fed into the pre-grown yeast cultures.

Cutinolytic enzyme assays

Spectrophotometric assay was used to determine the cutinase activity with p-nitrophenyl butyrate (PNB; Sigma, ST Louis, MO, USA) and p-nitrophenyl palmitate (PNP; Sigma, ST Louis, MO, USA) as substrates (Kim et al. 2003).

96-well microplate was used for the hydrolysis reactions at 30 °C for 4 min. Each well contained 200 μl of an enzyme/substrate solution (solution A), comprising 106.7 μl of phosphate buffer (0.1 M, pH 8.0), 13.3 μl of Triton X-100 (4 g l⁻¹), 13.3 μl of cell-free culture broth and 66.7 μl of substrate (PNB or PNP) solution. The PNB and PNP solutions were prepared with variable concentrations ranging from 0 to 1,000 mg l⁻¹. The initial velocity [i.e. the initial maximum rate of change in absorbance (OD_405nm s⁻¹)] was measured using a Molecular Devices EMax 96-well microplate reader (MDS Analytical Technologies, Toronto, Canada), and the enzyme-free solution A was used as a blank. As the eight wells in each of the 12 columns of the 96-well microplate contained an equal content of enzyme and substrate, the eight initial velocities measured for each column were used to calculate an average initial velocity for a specific reaction condition, resulting in an SD <5 %.

Enzyme activity of Pseudozyma japonica-Y7-09

Pseudozyma japonica-Y7-09 showed higher growth on YM medium fed with PCL film than on YM medium (Fig. 1). The substrate-specific hydrolysis and high accessibility to PNB are intrinsic properties of cutinase that have never been observed for hydrolysis by lipases and esterases (Kim et al. 2003). Isolation of natural cutinase from variable microbial sources would be easy using this distinct characteristic. The hydrolytic activities of cell-free supernatant from Pseudozyma japonica-Y7-09 culture broth on twop-nitrophenyl esters (PNB and PNP) were estimated for a range of substrate concentrations on time courses. The hydrolytic activities are defined as the initial maximum rate of hydrolysis at each substrate concentration as described previously (Kim et al. 2003; Seo et al. 2007). On the time course, the hydrolytic enzyme activity of the culture on PNB rapidly increased after 3 days and the maximum activity was at day 5. The initial maximum rate of PNB hydrolysis in YM and PCL medium was higher than that in YM medium (Fig. 2), whereas the hydrolytic activity on PNP was very low with the same range of substrate concentrations, even in YM and PCL medium. Therefore, it is likely that the cutinase production by Pseudozyma japonica-Y7-09 was induced by PCL. These results are similar to those obtained by Kim et al. 2003, Seo et al. 2007 using Pseudozyma jejuensis OL71.

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Fig. 1

Growth measured by culture turbidity (OD_600nm) in control (culture in YM) and culture in YM fed with PCL film (YM and PCL film)

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Fig. 2

Activity of enzyme secreted by Pseudozyma japonica-Y7-09 in yeast malt culture fed and that not fed with PCL film

During growth of Pseudozyma japonica-Y7-09 culture broth, the protein concentration rapidly increased after 3 days with a maximum concentration at 5 days similar to enzyme activity. Protein concentration recorded higher concentration in PCL feed YM medium than that in PCL free YM medium (Fig. 3).

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Fig. 3

Total protein concentration secreted by Pseudozyma japonica-Y7-09 in yeast malt culture fed and that not fed with PCL film

PCL degradation by Pseudozyma japonica-Y7-09

PCL dimers and trimers, products of enzymatic PCL degradation, are structurally similar to cutin degradation products that are known to be inducers of cutinase activity. This implies that oligomers of PCL may be natural inducers and substrates of cutinase (Nishida and Tokiwa 1993).

In the present study, PCL degradation was achieved in the conditions favouring the yeast strain’s growth in the YM medium in Erlenmeyer flasks as described above for 15 days. The weight loss of the degraded film was recorded and compared as a function of time. Under those conditions, partial degradation of PCL film (26.88 %) was recorded at the third day of incubation. That degradation reached to the maximum (93.33 %) at the 15 days (Figs. 4, ​,5).5). Weight loss was negligible in the same medium but free yeast cells (Figs. 4, ​,5).5). In an early study (Oda et al. 1995) isolated five strains of filamentous fungi of which Paecilomyceslilacinus was able to degrade 10 % of PCL after 10 days incubation. Xu et al. (2007) investigated the ability of several stock bacterial strains to degrade PCL. After 30 days of incubation, the weight loss of PCL film by the strain Lysinibacillus sp. 70,038 was 9 % while other strains showed less weight loss degradation.

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Fig. 4

a Degradation of PCL film after 0, 3, 5, 8, 12 and 15 days incubation time by Pseudozyma japonica-Y7-09 comparing with control. b Weight loss percentage of PCL film after 0, 3, 5, 8, 12 and 15 days incubation time by Pseudozyma japonica-Y7-09 comparing with control

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Fig. 5

Degradation of foam plastic by Pseudozyma japonica-Y7-09 incubated in yeast malt media for 30 days

We thank Dr. Kazunobu Matsushita (Yamaguchi University) for technical assistance.