# Poly-β-hydroxybutyrate  (PHB) Depolymerase from  Fusarium solani Thom

Authors: Srividya Shivakumar
Journal: Journal of Chemistry
 (2013)

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## Abstract

Fusarium solani Thom produced maximum PHB depolymerase by 48 h when grown in BHM containing 0.2%, w/v PHB, pH 8.0 at 25°C. Statistical optimization studies using Plackett Burman design of PHB depolymerase production yielded maximum PHB depolymerase activity after 2 days as against 4 days in the unoptimized conditions with a 2-fold increase in activity. Partial purification of the extracellular poly-β-hydroxybutyrate (PHB) depolymerase PHAZFus from F. solani Thom by two steps using ammonium sulphate (80% saturation) and affinity chromatography using concanavalin-A yielded 162.3-fold purity and 63% recovery of protein. The enzyme composed of a single polypeptide chain of 85 KDa, as determined by SDS-PAGE. The enzyme stained positive for glycoprotein by PAS staining. Optimum enzyme activity was detected at pH 7.0 and 55°C. The enzyme was stable at pH 7.0 and 55°C for 24 h with a residual activity of almost 85%. Paper chromatography revealed β-hydroxybutyrate monomer as the major end product of PHB hydrolysis. Complete inhibition of the enzyme by 1 mM HgCl2 (100%) indicated the importance of essential disulfide bonds (cystine residues) for enzyme activity or probably for maintaining the native enzyme structure.


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## Body

## 1. Introduction

Polyhydroxyalkanoates, PHAs, are versatile biopolyesters synthesized by numerous bacterial strains as intracellular storage compounds of carbon and energy. These polyesters can be divided primarily into two classes of short-chain-length (SCL) PHAs and medium-chain-length (MCL) PHAs, according to the carbon-chain-length of constituents: SCL-PHAs consist ofR-hydroxyalkanoates of C3–C5 and MCL-PHAs are comprised of R-hydroxyalkanoates of C6-C14. Poly-3-hydroxybutyrate (PHB), a representative SCL-PHA, is a promising material for use as a renewable and biodegradable plastic [1].It has been reported that PHB-degrading bacteria are distributed widely in the natural environment, and several extracellular PHB depolymerases with differing biochemical properties have been isolated from various bacterial origins [2, 3]. However, the ability to degrade SCL-PHA is not restricted to bacteria, and some filamentous fungi also play an important role in the extracellular degradation of SCL-PHAs such as PHB and its copolyesters with 3-hydroxyvalerate [4]. Many reports have been published on the fungal degradation of SCL-PHAs in the environment, however reports on the properties of the PHB depolymerases from fungi are relatively rare. Only few fungal PHB depolymerases have been purified and partially characterized to date [5–12]. Therefore, the biochemical properties of fungal PHB depolymerases are not well documented in comparison to those of bacterial PHB depolymerases.The ever-increasing human demand for materials leads to a concomitant increase in waste production. The fact that soil   serves as major landfill, it also means that the new generation of biosynthetic plastics will probably have to be degraded there as well. The fungal biomass in soils generally exceeds the bacterial biomass and thus it is likely that fungi may play a considerable role in degrading polyesters, just as they predominantly perform the decomposition of organic matter in the soil ecosystem. However, in contrast to bacterial polyester degradation, which has been extensively investigated, the microbiological and environmental aspects of fungal degradation of polyesters are unclear [13]. Many PHA-degrading fungi have been identified [4, 14]. Soil-containing environments are the habitats from which the largest numbers of fungal PHB degraders are found [15].  To our knowledge there are only a few reports on the biochemical characteristics of fungal PHB depolymerases and no report so far on statistical optimization of medium components for production of any fungal PHB depolymerase.Optimizing the parameters by statistical method reduces the time and expense; otherwise if required, conventional one-factor-at-a-time approach is used. Several statistical methods are available for optimization experiments [16]. Plackett and Burman’s statistical method is one of such approaches involving a two-level fractional factorial saturated design that uses only k+1 treatment combinations to estimate the main effects of k factors independently, assuming that all interactions are negligible [17]. Saturated designs are used in the early stages of experimentation to screen out unimportant factors from among a large number of possible factors. In full factorial designs, the number of factors increases exponentially leading to an unmanageable number of experiments [18]. Hence, fractional factorial design like Plackett-Burman becomes a method of choice for initial screening of medium components.The present paper describes the production, purification process and some of the properties of the PHB depolymerase obtained from a fungal isolate,Fusarium solaniThom. The exceptional properties of this enzyme are emphasized and compared with those of other fungal depolymerases.


## 2. Experimental

### 2.1. PHB

PHB was obtained as a kind gift from Biomer Inc., Germany. The molecular weight of PHB was 470,000 g/mol. All experiments were performed using PHB powder.


### 2.2. Isolation of a SCL-PHA-Degrading Fungus

A SCL-PHA-degrading filamentous fungus from a wastewater sample was isolated by pure culturing a colony with high depolymerase activity among fungi grown on a mineral salt agar medium [7]  containing PHB as the sole carbon source.


### 2.3. Optimization of the Medium Using Statistically Based Plackett-Burman Experimental Design

The Plackett-Burman experimental design, a fractional factorial design [17, 19] was used in this research to reflect the relative importance of various environmental factors on PHB degradation and PHB depolymerase activity in liquid cultures.


### 2.4. Production of the PHB Depolymerase from the Isolate

The isolate was cultivated in mineral salts agar medium at 30°C for 3-4 days as described earlier [12].  The culture supernatant was filtered using Whatmann filter paper no. 1 and used as enzyme source.


### 2.5. Protein Estimation

Protein concentrations were measured by Bradford's method using bovine serum albumin as the standard [20].


### 2.6. Enzyme Assay

The PHB depolymerase assay was carried out by adding the enzyme to 100 mM  sodium citrate buffer (pH 6.0) containing 300μg of PHB at 55°C for 1 h [11].


### 2.7. Purification of PHB Depolymerase

The extracellular PHB depolymerase of the fungus was purified from the culture filtrate by (NH4)2SO4 precipitation (80% saturation), followed by affinity chromatography using concanavalin-A [12]. The fractions collected were assayed for enzyme activity and protein content by the method mentioned above.


### 2.8. Molecular Weight Determination

Electrophoresis (10%, w/v sodium dodecyl sulfate polyacrylamide gel electrophoresis) was carried out to measure the molecular weight according to Yu [19].  Phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.5 kDa) were used as standard molecular weight markers. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250.


### 2.9. Glycoprotein Staining

Carbohydrate staining of glycoprotein in SDS-PAGE gel was carried out with fuchsin sulfate after periodate oxidation [21].


### 2.10. Effects of Temperature and pH on the Enzyme Activity

To find out the effect of temperature on PHB depolymerase activity, enzyme activity was measured at different temperatures in the range of 25°C to 60°C at optimum pH. Temperature stability was  measured by incubating the enzyme in standard reaction solution without PHB for 90 min at 25~60°C, followed by enzyme assay. The optimum pH for enzyme activity was determined by measuring the activity in each of the buffers containing PHB and enzyme. To examine the pH stability of the enzyme, different buffers containing enzyme without substrate were incubated at 25°C for 2 h, and then controlled to have the optimum pH in order to measure the residual activity of the enzyme [12].


### 2.11. Determination ofKm and Vmax

A PHB stock solution (3 mg/mL) was used to prepare varying PHB (substrate) concentrations from 200–2000μg/mL, making up the volume to 2 mL with buffer (optimal assay pH). Using the data, a double reciprocal plot (Lineweaver-Burke plot) was plotted and the value of Km was determined [12].


### 2.12. Substrate Specificity

To examine the ability of the PHB depolymerase to degrade other polymers, PCL and copolymer of poly-β-hydroxybutyrate (5%) were used. The standard assay under optimal conditions was performed for the enzyme extract using P (HB-co-HV) (5%), PCL (5%)  as the substrate instead of PHB. The absorbance was read at 660 nm against a blank [12].


### 2.13. Esterase Activity

Esterase activity was assayed in 2 mL of 100 mM sodium citrate buffer, pH 6.0, usingp-nitrophenyl alkanoates-para-nitrophenyl acetate (PNPA), para-nitrophenyl butyrate (PNPB), and para-nitrophenyl decanoate (PNPD) incubated at 37°C for 10 min. The reaction mixtures contained 50 μL of a 10 mM solution of the substrates in ethanol and 10 μL of the enzyme solution [12]. Polycaprolactone (PCL; MWavg, 42,500) and other substrates were purchased  from the Aldrich Chemical Co. Suspensions of PCL were prepared as previously described [22].


### 2.14. Effect of Inhibitors

The inhibitory effect of various chemical reagents on enzyme activity was measured as follows: the reaction mixture (1.97 mL) containing 1 mL of the enzyme solution, reagent, and 100 mM sodium citrate buffer (pH 6.0) was initially pre-incubated for 1 h at 37°C, with the enzymatic reaction subsequently started by adding 2 mL of the PHB substrate. All the inhibitors were purchased from Sigma (St. Louis, MO, USA) [22].


### 2.15. Identification of PHB Hydrolysis End Products

One milligram  of PHB was incubated in 1 mL 100 mM sodium citrate buffer (pH 6.0) at 50°C in the presence of 10μg purified PHB depolymerase for 4–6 h. After incubation, the reaction was stopped with 0.1 N HCl, reaction mixture acidified to pH 4.0, and the aqueous end product was repeatedly extracted with equal volumes of diethyl ether in presence of 1 N NaCl in a separating funnel. The ether extract was collected and air-dried. The concentrated extract was detected for the presence of aqueous PHB hydrolysis products by paper chromatography using 2° Butanol: H2O: 28% NH4OH in the ratio 1,000 : 200 : 3 and Bromocresol purple (0.1%, w/v in distilled water) as developer. The monomer of complete PHB hydrolysis-β hydroxybutyrate (Sigma) was spotted as reference. The paper was observed for yellow spots against purple background.


## 2.1. PHB

PHB was obtained as a kind gift from Biomer Inc., Germany. The molecular weight of PHB was 470,000 g/mol. All experiments were performed using PHB powder.


## 2.2. Isolation of a SCL-PHA-Degrading Fungus

A SCL-PHA-degrading filamentous fungus from a wastewater sample was isolated by pure culturing a colony with high depolymerase activity among fungi grown on a mineral salt agar medium [7]  containing PHB as the sole carbon source.


## 2.3. Optimization of the Medium Using Statistically Based Plackett-Burman Experimental Design

The Plackett-Burman experimental design, a fractional factorial design [17, 19] was used in this research to reflect the relative importance of various environmental factors on PHB degradation and PHB depolymerase activity in liquid cultures.


## 2.4. Production of the PHB Depolymerase from the Isolate

The isolate was cultivated in mineral salts agar medium at 30°C for 3-4 days as described earlier [12].  The culture supernatant was filtered using Whatmann filter paper no. 1 and used as enzyme source.


## 2.5. Protein Estimation

Protein concentrations were measured by Bradford's method using bovine serum albumin as the standard [20].


## 2.6. Enzyme Assay

The PHB depolymerase assay was carried out by adding the enzyme to 100 mM  sodium citrate buffer (pH 6.0) containing 300μg of PHB at 55°C for 1 h [11].


## 2.7. Purification of PHB Depolymerase

The extracellular PHB depolymerase of the fungus was purified from the culture filtrate by (NH4)2SO4 precipitation (80% saturation), followed by affinity chromatography using concanavalin-A [12]. The fractions collected were assayed for enzyme activity and protein content by the method mentioned above.


## 2.8. Molecular Weight Determination

Electrophoresis (10%, w/v sodium dodecyl sulfate polyacrylamide gel electrophoresis) was carried out to measure the molecular weight according to Yu [19].  Phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.5 kDa) were used as standard molecular weight markers. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250.


## 2.9. Glycoprotein Staining

Carbohydrate staining of glycoprotein in SDS-PAGE gel was carried out with fuchsin sulfate after periodate oxidation [21].


## 2.10. Effects of Temperature and pH on the Enzyme Activity

To find out the effect of temperature on PHB depolymerase activity, enzyme activity was measured at different temperatures in the range of 25°C to 60°C at optimum pH. Temperature stability was  measured by incubating the enzyme in standard reaction solution without PHB for 90 min at 25~60°C, followed by enzyme assay. The optimum pH for enzyme activity was determined by measuring the activity in each of the buffers containing PHB and enzyme. To examine the pH stability of the enzyme, different buffers containing enzyme without substrate were incubated at 25°C for 2 h, and then controlled to have the optimum pH in order to measure the residual activity of the enzyme [12].


## 2.11. Determination ofKm and Vmax

A PHB stock solution (3 mg/mL) was used to prepare varying PHB (substrate) concentrations from 200–2000μg/mL, making up the volume to 2 mL with buffer (optimal assay pH). Using the data, a double reciprocal plot (Lineweaver-Burke plot) was plotted and the value of Km was determined [12].


## 2.12. Substrate Specificity

To examine the ability of the PHB depolymerase to degrade other polymers, PCL and copolymer of poly-β-hydroxybutyrate (5%) were used. The standard assay under optimal conditions was performed for the enzyme extract using P (HB-co-HV) (5%), PCL (5%)  as the substrate instead of PHB. The absorbance was read at 660 nm against a blank [12].


## 2.13. Esterase Activity

Esterase activity was assayed in 2 mL of 100 mM sodium citrate buffer, pH 6.0, usingp-nitrophenyl alkanoates-para-nitrophenyl acetate (PNPA), para-nitrophenyl butyrate (PNPB), and para-nitrophenyl decanoate (PNPD) incubated at 37°C for 10 min. The reaction mixtures contained 50 μL of a 10 mM solution of the substrates in ethanol and 10 μL of the enzyme solution [12]. Polycaprolactone (PCL; MWavg, 42,500) and other substrates were purchased  from the Aldrich Chemical Co. Suspensions of PCL were prepared as previously described [22].


## 2.14. Effect of Inhibitors

The inhibitory effect of various chemical reagents on enzyme activity was measured as follows: the reaction mixture (1.97 mL) containing 1 mL of the enzyme solution, reagent, and 100 mM sodium citrate buffer (pH 6.0) was initially pre-incubated for 1 h at 37°C, with the enzymatic reaction subsequently started by adding 2 mL of the PHB substrate. All the inhibitors were purchased from Sigma (St. Louis, MO, USA) [22].


## 2.15. Identification of PHB Hydrolysis End Products

One milligram  of PHB was incubated in 1 mL 100 mM sodium citrate buffer (pH 6.0) at 50°C in the presence of 10μg purified PHB depolymerase for 4–6 h. After incubation, the reaction was stopped with 0.1 N HCl, reaction mixture acidified to pH 4.0, and the aqueous end product was repeatedly extracted with equal volumes of diethyl ether in presence of 1 N NaCl in a separating funnel. The ether extract was collected and air-dried. The concentrated extract was detected for the presence of aqueous PHB hydrolysis products by paper chromatography using 2° Butanol: H2O: 28% NH4OH in the ratio 1,000 : 200 : 3 and Bromocresol purple (0.1%, w/v in distilled water) as developer. The monomer of complete PHB hydrolysis-β hydroxybutyrate (Sigma) was spotted as reference. The paper was observed for yellow spots against purple background.


## 3. Results and Discussion

### 3.1. Microorganism: Identification and Characterization

Fungal species identified asFusarium solani Thom (Fungal identification service, Agarkar Research Institute, Pune) based on morphotaxonomy, was used in this work.


### 3.2. Optimization of PHB Depolymerase Production by Plackett-Burman Design

The independent variables examined in Plackett-Burman design and their levels are presented in Table1 [23].Table 1
Degree of positive or negative effect of independent variables onF.  solani Thom PHB depolymerase according to the levels in the Plackett-Burman experimental design.
Variable
Symbol
(−) Level
(0) Level
(+) Level
Main effect
T value
Significance level
Time (h)
a
48
72
96
0.6667
−3.89
NS
pH
b
4
6
8
0.1422
−0.853
NS
Temperature (°C)
c
25
30
35
−0.5687
4.32
S
Inoculum size (%, v/v)
d
2
4
6
0.0688
−0.397
NS
PHB (g/L)
e
1
2
3
0
0
NS
Glucose (g/L)
f
5
0
10
0
0
S
Volume/flask (mL)
g
20
50
100
−0.2447
1.89
S
Nitrogen (g/L)
h
0.5
1.0
1.5
−0.02
3.761
S
KH2PO4 (g/L)
i
0.1
0.7
1.3
−0.0512
0.57
NS
Bold type indicates positive effect.
Significant (S); nonsignificant (NS).t0.1=1.875; t0.05=2.365.The design was applied with different fermentation conditions. The main effect of each variable upon PHB degradation and the production of PHB depolymerase enzyme was estimated and presented in Figure1.Figure 1
Elucidation of fermentation factors affecting PHB depolymerase activity ofF. solani Thom.Within the range of the tested levels of variables, pH, time of incubation, and  inoculum size affected activity at high levels (Figure1). On the other hand, other variables showed effects on PHB depolymerase activity at low levels. Different concentrations of PHB did not show any effect on the enzyme activity in the range tested. Presence of glucose showed repressive effect on the enzyme activity suggesting the catabolic repression effect of glucose on PHB depolymerase production. Of all the factors tested, time of incubation and temperature showed profound effect on PHB depolymerase activity at high and low levels, respectively, indicating the importance of these two  factors in production of the enzyme.According to the data obtained from the Plackett-Burman experimental results, KH2PO4, and NH4Cl should be added in the lowest concentration and the medium predicted to be near optimum should be of the following composition (g/L): PHB, 2; MgSO4·7H2O, 0.2; NH4Cl, 1;   KH2PO4, 0.1; CaCl2, 0.02;  FeCl3, 0.05. Fungal spores were inoculated with 6% (v/v) inoculum of 5-day-old culture in a total culture volume of 25 mL BHM and incubated at pH 8.0 and 25°C. Under optimized culture conditions, maximum PHB depolymerase activity was achieved after 2 days as against 4 days in the unoptimized conditions with a 2-fold increase in activity (Figures 2 and 3).Figure 2
Plate showing PHB clearance byF. solani Thom.Figure 3
Time course production of PHB depolymerase byF. solani Thom.In order to optimize enzyme production, the Plackett-Burman design was applied, which has been demonstrated as an efficient approach to screen for medium components and/or factors affecting PHB degradation. Increasing pH level, proved to enhance polymer hydrolysis. On the contrary, increasing NH4Cl and KH2PO4 level negatively affected enzyme activity. PHB concentration at the tested levels did not show profound influence on the enzyme activity. Supplementation of glucose negatively affected PHB depolymerase production by this isolate suggesting catabolite repression by glucose. Our results are in agreement to all other known PHB bacterial depolymerases, which are repressed in the presence of a soluble carbon source. In accordance with our results are those reported for Pseudomonas lemoignei,[24, 25] which produced polymerase maximally during growth on succinate. This result is in good agreement with a previous investigation which demonstrated the importance of carbon sources in the growth medium for enzyme production as the rate of polymer degradation was influenced by the degree and availability of secondary carbon and by the initial carbon in the liquid medium. In contrast to this observation, the PHB depolymerase of Nocardiopsis aegyptia was found to be  constitutive [23].


### 3.3. Purification of PHB Depolymerase

Purification of the enzyme by two steps using ammonium sulphate precipitation (80% saturation) and affinity chromatography using concanavalin-A yielded 162.31-fold purity with 63% recovery of the protein (Table2). PHB depolymerases from different systems have been purified using different multistep methods. According to the literature, the yield of PHB depolymerase after purification was 66% from P. funiculosum; 27% from  A. faecalis; 42% from Pseudomonassp.; 66% from A. fumigatus; 73.5% from Aureobacterium saperdae; 61% from P. simplicissimum and 86.11% from A. fumigatus Pdf1.  The increases of the enzyme activity were 4.5-fold for P. funiculosum; 1.5-fold for A. faecalis; and 5.67-fold for Pseudomonassp.; 2.4-fold in P. simplicissimum  and 33.56-fold in A. fumigatus Pdf1 [5, 7, 9, 11, 26–28].Table 2
Purification of PHB depolymerase ofF.  solani Thom.
Purification step
Enzyme activity(U/mL)
Total enzyme activity (U)
Protein (mg/mL)
Total protein (mg)
Specific activity (U/mg)
Purification fold
Activity recovery (%)
Crude enzyme (1000 mL)
5.0
5000
0.260
160
18.75
1.0
100
NH4SO4 ppt, dialysed (9.0 mL)
410
3690
2.5
28.8
128.125
6.83
73.8
Affinity chromatography (9 mL)
350
3150
0.115
1.035
3043.4
162.31
63


### 3.4. Molecular Weight Determination

The purity and homogeneity of the enzyme was confirmed by (Figure4(a), Lanes 2 and 3) protein staining. On performing activity staining on 2.5% (w/v) PHB plates, a zone of clearance was observed with different concentrations of the pure enzyme (Figure 4(b)). The molecular weight of F. solani PHB depolymerase determined here is in agreement   to that of the PHB depolymerase obtained from P. funiculosum; P. pinophilum; P. simplicissimum and A. fumigatus Pdf1 and P. citrinum[5–7, 9, 12]; bacterial PHB depolymerase [24, 26–28] all of which showed a single polypeptide of different molecular weights. The PHB depolymerase of A. faecalis AE122 and   P. citrinum S2 are the only depolymerases reported with unusually high apparent Mr of 96 KDa and  240 KDa [12, 27].(a) SDS denatured PAGE of standard marker (Lane 1), purified PHB depolymerase by ammonium sulphate precipitation (Lane 2), and affinity chromatography (Lane 3). (b) Activity staining of PHB depolymerase ofF. solaniThom on PHB plate.
(a)
(b)


### 3.5.  Glycoprotein Staining

On subjecting the PHB depolymerase ofF. solani to glycoprotein staining by PAS method after SDS PAGE (Figure 5) revealed glycosylation. Carbohydrates were not detected in the PHB depolymerase either from A. fumigatus or from A. saperdae [11, 28]. However, the bacterial PHB depolymerase from Pseudomonas lemoignei[24]; and fungal PHB depolymerase fromP. funiculosum[5]; P. simplicissimum[9]; A. fumigatus Pdf1 [7], and P. citrinum S2 [12] were glycosylated. The composition and function of the carbohydrate moiety of the glycosylated PHB depolymerase and other proteins studied so far are unknown. The cellulases of Trichoderma reeseiand Cellulomonas fimiare known to be glycosylated in the proline/threonine rich region and an extracellular glucoamylase from Aspergillus niger is also glycosylated in the C-terminal region of the protein. A functional role of the carbohydrate moiety in binding the substrate of cellulases has been proposed. However, glycosylation is not essential for activity but may prove the resistance of the exoenzyme to elevated temperature and/or hydrolytic cleavage by proteases of competing microorganisms [29].Figure 5
Glycoprotein staining of PHB depolymerase by PAS method. Standard molecular weight marker (Coomassie stained) (Lane 1); purified PHB depolymerase by ammonium sulphate precipitation (Lane 2),  and affinity chromatography (Lane 3) after PAS method.


### 3.6. Effects of Temperature and pH on the Enzyme Activity

The optimum pH of the PHB depolymerase from fungi [24, 26–28] is  in the range of 7.5~9.5 depending on the strain. The PHB depolymerase enzyme of F. solani was most active at pH 7.0 as shown in Figure 6. pH stability of  F. solani  PHB depolymerase at pH 7.0, 55°C showed stability over a period of 24 h with a residual activity of almost 85% (Figure 7). The PHB depolymerase of A. saperdae [28] was completely inactivated at pH 1.5 in 2 hours and that of P. lilacinus[10] maintained the activity only in the range of pH 4.0 to 6.0. Accordingly, it was concluded that the PHB depolymerase of F. solani was very stable under neutral  conditions.Figure 6
pH optima ofF. solani PHB depolymerase.Figure 7
pH stability ofF. solani PHB depolymerase.The enzyme exhibited the highest activity at 55°C and was stable at 55°C for 1 h with 84% residual activity  (Figures8 and 9), indicating that the enzyme was thermally as stable as other PHB depolymerases reported [5, 11, 27, 30]. The PHB depolymerase produced by PHB decomposing organisms has high activity and stability at high temperatures.Figure 8
Temperature optima ofF. solani PHB depolymerase.Figure 9
Temperature stability ofF. solani PHB depolymerase.


### 3.7. Determination ofKm and Vmax

TheKm values were determined by plotting a double reciprocal plot (Figure 10). The Km and Vmax values are 100 μg/mL and 50 μg/min, respectively.Figure 10
Determination ofKm and  Vmax of F. solani PHB depolymerase by double reciprocal plot.


### 3.8. Effect of Inhibitors

Inhibitors are indicative of the various functional groups present in the active site of an enzyme. Effect of inhibitors on the activity of the enzyme was investigated in order to identify the active sites in the PHB depolymerase ofF. solani (Figure 11). The activity of F. solani PHB depolymerase seemed to be unaffected by N-p-tosyl-L-lysinechloromethyl ketone (TLCK), which should transform the histidine groups. The PHB depolymerases of P. funiculosum  and A. saperdae also exhibited 100% activity in the presence of 10 mM TLCK [5, 28]. Complete inhibition of the F. solani enzyme by 1 mM HgCl2 (100%), indicates the importance of essential disulfide bonds (cystine residues) for enzyme activity or probably for maintaining the native enzyme structure. Partial inhibition of the enzyme  by  PMSF (10 mM), DAN (10 mM) and N-ethylmaleimide (10 mM), indicating less importance of serine residue, carboxyl group, tyrosine, and sulfhydryl groups  in the active site of the enzyme respectively [28]. In contrast, P. simplicissimumLAR 13 PHB depolymerase was not affected by N-acetylimidazole. Phenylmethylsulfonyl fluoride (PMSF) is known as an inhibitor of serine residues [5, 24, 30]. The PHB depolymerase of A. saperdae was partially inactivated by 10 mM PMSF [28], and that of P. lemoignei [24] and Agrobacteriumsp. [31] was completely inhibited by 1 mM PMSF. However, the PHB depolymerase of P. simplicissimumLAR 13 showed 58% activity in the presence of 10 mM PMSF [9]. The inhibitor studies clearly shows distinct nature of F. solani PHB depolymerase active site as compared to the PHB depolymerases reported till date.Figure 11
Effect of inhibitors onF. solani PHB depolymerase.


### 3.9. Substrate Specificity

Of the PNP-alkanoates tested, PNP-decanoate and copolymer P(HB-co-HV) were hydrolyzed efficiently byF. solani PHB depolymerase and the esterase activity for the substrate was measured at 180 units, respectively (Table 3), indicating the true depolymerase nature of the enzyme.  The enzyme also showed appreciable activity with other PNP-alkanoates like PNP-acetate (PNPA) and PNP-butyrate (PNPB) which is in agreement with the  PHB depolymerase of E. minima [8] and A. fumigatus Pdf1 [7]. Absence of activity with PCL indicates that the enzyme is not a cutinase [32].Table 3
Substrate specificities ofF.  solani PHB depolymerase on different polymers.
Substrates
Activity (Units/mL/min)
PNPA
a
0.6
PNPB
b
3.2
PNPD
c
180
PCL
d
0
P(HB-Co-HV)e
180
P
aolynitrophenyl acetate; pbolynitrophenyl butyrate; pcolynitrophenyl  decanoate; pdolycaprolactone; peolymer of polyhydroxybutyrate and hydroxyvalerate.


### 3.10. Identification of PHB Hydrolysis End-Products

Identification of the aqueous end-products of PHB depolymerase reaction by paper chromatography revealedβ-hydroxybutyrate monomer as major end product of PHB hydrolysis (Figure 12). Though high-resolution analysis or HPLC/LC-MS analysis is required to confirm the above observations, this preliminary observation by paper chromatography indicated mainly monomers as the aqueous end-product of PHB hydrolysis by Penicilliumsp. S2 PHB depolymerase as in the case of Comamonas sp[33] and P. picketti[30], as against the depolymerase of A. faecalis T1,A. faecalisAE122, and P. lemoigneiwhich hydrolyze PHB mainly to the dimeric and trimeric ester of hydroxybutyrate [8, 10, 24, 27].Figure 12
Identification of PHB hydrolysis end products by PHB depolymerase ofF. solani. Lane 1—β-hydroxybutyrate standard, Lane 2—end-product of PHB depolymerase reaction.


## 3.1. Microorganism: Identification and Characterization

Fungal species identified asFusarium solani Thom (Fungal identification service, Agarkar Research Institute, Pune) based on morphotaxonomy, was used in this work.


## 3.2. Optimization of PHB Depolymerase Production by Plackett-Burman Design

The independent variables examined in Plackett-Burman design and their levels are presented in Table1 [23].Table 1
Degree of positive or negative effect of independent variables onF.  solani Thom PHB depolymerase according to the levels in the Plackett-Burman experimental design.
Variable
Symbol
(−) Level
(0) Level
(+) Level
Main effect
T value
Significance level
Time (h)
a
48
72
96
0.6667
−3.89
NS
pH
b
4
6
8
0.1422
−0.853
NS
Temperature (°C)
c
25
30
35
−0.5687
4.32
S
Inoculum size (%, v/v)
d
2
4
6
0.0688
−0.397
NS
PHB (g/L)
e
1
2
3
0
0
NS
Glucose (g/L)
f
5
0
10
0
0
S
Volume/flask (mL)
g
20
50
100
−0.2447
1.89
S
Nitrogen (g/L)
h
0.5
1.0
1.5
−0.02
3.761
S
KH2PO4 (g/L)
i
0.1
0.7
1.3
−0.0512
0.57
NS
Bold type indicates positive effect.
Significant (S); nonsignificant (NS).t0.1=1.875; t0.05=2.365.The design was applied with different fermentation conditions. The main effect of each variable upon PHB degradation and the production of PHB depolymerase enzyme was estimated and presented in Figure1.Figure 1
Elucidation of fermentation factors affecting PHB depolymerase activity ofF. solani Thom.Within the range of the tested levels of variables, pH, time of incubation, and  inoculum size affected activity at high levels (Figure1). On the other hand, other variables showed effects on PHB depolymerase activity at low levels. Different concentrations of PHB did not show any effect on the enzyme activity in the range tested. Presence of glucose showed repressive effect on the enzyme activity suggesting the catabolic repression effect of glucose on PHB depolymerase production. Of all the factors tested, time of incubation and temperature showed profound effect on PHB depolymerase activity at high and low levels, respectively, indicating the importance of these two  factors in production of the enzyme.According to the data obtained from the Plackett-Burman experimental results, KH2PO4, and NH4Cl should be added in the lowest concentration and the medium predicted to be near optimum should be of the following composition (g/L): PHB, 2; MgSO4·7H2O, 0.2; NH4Cl, 1;   KH2PO4, 0.1; CaCl2, 0.02;  FeCl3, 0.05. Fungal spores were inoculated with 6% (v/v) inoculum of 5-day-old culture in a total culture volume of 25 mL BHM and incubated at pH 8.0 and 25°C. Under optimized culture conditions, maximum PHB depolymerase activity was achieved after 2 days as against 4 days in the unoptimized conditions with a 2-fold increase in activity (Figures 2 and 3).Figure 2
Plate showing PHB clearance byF. solani Thom.Figure 3
Time course production of PHB depolymerase byF. solani Thom.In order to optimize enzyme production, the Plackett-Burman design was applied, which has been demonstrated as an efficient approach to screen for medium components and/or factors affecting PHB degradation. Increasing pH level, proved to enhance polymer hydrolysis. On the contrary, increasing NH4Cl and KH2PO4 level negatively affected enzyme activity. PHB concentration at the tested levels did not show profound influence on the enzyme activity. Supplementation of glucose negatively affected PHB depolymerase production by this isolate suggesting catabolite repression by glucose. Our results are in agreement to all other known PHB bacterial depolymerases, which are repressed in the presence of a soluble carbon source. In accordance with our results are those reported for Pseudomonas lemoignei,[24, 25] which produced polymerase maximally during growth on succinate. This result is in good agreement with a previous investigation which demonstrated the importance of carbon sources in the growth medium for enzyme production as the rate of polymer degradation was influenced by the degree and availability of secondary carbon and by the initial carbon in the liquid medium. In contrast to this observation, the PHB depolymerase of Nocardiopsis aegyptia was found to be  constitutive [23].


## 3.3. Purification of PHB Depolymerase

Purification of the enzyme by two steps using ammonium sulphate precipitation (80% saturation) and affinity chromatography using concanavalin-A yielded 162.31-fold purity with 63% recovery of the protein (Table2). PHB depolymerases from different systems have been purified using different multistep methods. According to the literature, the yield of PHB depolymerase after purification was 66% from P. funiculosum; 27% from  A. faecalis; 42% from Pseudomonassp.; 66% from A. fumigatus; 73.5% from Aureobacterium saperdae; 61% from P. simplicissimum and 86.11% from A. fumigatus Pdf1.  The increases of the enzyme activity were 4.5-fold for P. funiculosum; 1.5-fold for A. faecalis; and 5.67-fold for Pseudomonassp.; 2.4-fold in P. simplicissimum  and 33.56-fold in A. fumigatus Pdf1 [5, 7, 9, 11, 26–28].Table 2
Purification of PHB depolymerase ofF.  solani Thom.
Purification step
Enzyme activity(U/mL)
Total enzyme activity (U)
Protein (mg/mL)
Total protein (mg)
Specific activity (U/mg)
Purification fold
Activity recovery (%)
Crude enzyme (1000 mL)
5.0
5000
0.260
160
18.75
1.0
100
NH4SO4 ppt, dialysed (9.0 mL)
410
3690
2.5
28.8
128.125
6.83
73.8
Affinity chromatography (9 mL)
350
3150
0.115
1.035
3043.4
162.31
63


## 3.4. Molecular Weight Determination

The purity and homogeneity of the enzyme was confirmed by (Figure4(a), Lanes 2 and 3) protein staining. On performing activity staining on 2.5% (w/v) PHB plates, a zone of clearance was observed with different concentrations of the pure enzyme (Figure 4(b)). The molecular weight of F. solani PHB depolymerase determined here is in agreement   to that of the PHB depolymerase obtained from P. funiculosum; P. pinophilum; P. simplicissimum and A. fumigatus Pdf1 and P. citrinum[5–7, 9, 12]; bacterial PHB depolymerase [24, 26–28] all of which showed a single polypeptide of different molecular weights. The PHB depolymerase of A. faecalis AE122 and   P. citrinum S2 are the only depolymerases reported with unusually high apparent Mr of 96 KDa and  240 KDa [12, 27].(a) SDS denatured PAGE of standard marker (Lane 1), purified PHB depolymerase by ammonium sulphate precipitation (Lane 2), and affinity chromatography (Lane 3). (b) Activity staining of PHB depolymerase ofF. solaniThom on PHB plate.
(a)
(b)


## 3.5.  Glycoprotein Staining

On subjecting the PHB depolymerase ofF. solani to glycoprotein staining by PAS method after SDS PAGE (Figure 5) revealed glycosylation. Carbohydrates were not detected in the PHB depolymerase either from A. fumigatus or from A. saperdae [11, 28]. However, the bacterial PHB depolymerase from Pseudomonas lemoignei[24]; and fungal PHB depolymerase fromP. funiculosum[5]; P. simplicissimum[9]; A. fumigatus Pdf1 [7], and P. citrinum S2 [12] were glycosylated. The composition and function of the carbohydrate moiety of the glycosylated PHB depolymerase and other proteins studied so far are unknown. The cellulases of Trichoderma reeseiand Cellulomonas fimiare known to be glycosylated in the proline/threonine rich region and an extracellular glucoamylase from Aspergillus niger is also glycosylated in the C-terminal region of the protein. A functional role of the carbohydrate moiety in binding the substrate of cellulases has been proposed. However, glycosylation is not essential for activity but may prove the resistance of the exoenzyme to elevated temperature and/or hydrolytic cleavage by proteases of competing microorganisms [29].Figure 5
Glycoprotein staining of PHB depolymerase by PAS method. Standard molecular weight marker (Coomassie stained) (Lane 1); purified PHB depolymerase by ammonium sulphate precipitation (Lane 2),  and affinity chromatography (Lane 3) after PAS method.


## 3.6. Effects of Temperature and pH on the Enzyme Activity

The optimum pH of the PHB depolymerase from fungi [24, 26–28] is  in the range of 7.5~9.5 depending on the strain. The PHB depolymerase enzyme of F. solani was most active at pH 7.0 as shown in Figure 6. pH stability of  F. solani  PHB depolymerase at pH 7.0, 55°C showed stability over a period of 24 h with a residual activity of almost 85% (Figure 7). The PHB depolymerase of A. saperdae [28] was completely inactivated at pH 1.5 in 2 hours and that of P. lilacinus[10] maintained the activity only in the range of pH 4.0 to 6.0. Accordingly, it was concluded that the PHB depolymerase of F. solani was very stable under neutral  conditions.Figure 6
pH optima ofF. solani PHB depolymerase.Figure 7
pH stability ofF. solani PHB depolymerase.The enzyme exhibited the highest activity at 55°C and was stable at 55°C for 1 h with 84% residual activity  (Figures8 and 9), indicating that the enzyme was thermally as stable as other PHB depolymerases reported [5, 11, 27, 30]. The PHB depolymerase produced by PHB decomposing organisms has high activity and stability at high temperatures.Figure 8
Temperature optima ofF. solani PHB depolymerase.Figure 9
Temperature stability ofF. solani PHB depolymerase.


## 3.7. Determination ofKm and Vmax

TheKm values were determined by plotting a double reciprocal plot (Figure 10). The Km and Vmax values are 100 μg/mL and 50 μg/min, respectively.Figure 10
Determination ofKm and  Vmax of F. solani PHB depolymerase by double reciprocal plot.


## 3.8. Effect of Inhibitors

Inhibitors are indicative of the various functional groups present in the active site of an enzyme. Effect of inhibitors on the activity of the enzyme was investigated in order to identify the active sites in the PHB depolymerase ofF. solani (Figure 11). The activity of F. solani PHB depolymerase seemed to be unaffected by N-p-tosyl-L-lysinechloromethyl ketone (TLCK), which should transform the histidine groups. The PHB depolymerases of P. funiculosum  and A. saperdae also exhibited 100% activity in the presence of 10 mM TLCK [5, 28]. Complete inhibition of the F. solani enzyme by 1 mM HgCl2 (100%), indicates the importance of essential disulfide bonds (cystine residues) for enzyme activity or probably for maintaining the native enzyme structure. Partial inhibition of the enzyme  by  PMSF (10 mM), DAN (10 mM) and N-ethylmaleimide (10 mM), indicating less importance of serine residue, carboxyl group, tyrosine, and sulfhydryl groups  in the active site of the enzyme respectively [28]. In contrast, P. simplicissimumLAR 13 PHB depolymerase was not affected by N-acetylimidazole. Phenylmethylsulfonyl fluoride (PMSF) is known as an inhibitor of serine residues [5, 24, 30]. The PHB depolymerase of A. saperdae was partially inactivated by 10 mM PMSF [28], and that of P. lemoignei [24] and Agrobacteriumsp. [31] was completely inhibited by 1 mM PMSF. However, the PHB depolymerase of P. simplicissimumLAR 13 showed 58% activity in the presence of 10 mM PMSF [9]. The inhibitor studies clearly shows distinct nature of F. solani PHB depolymerase active site as compared to the PHB depolymerases reported till date.Figure 11
Effect of inhibitors onF. solani PHB depolymerase.


## 3.9. Substrate Specificity

Of the PNP-alkanoates tested, PNP-decanoate and copolymer P(HB-co-HV) were hydrolyzed efficiently byF. solani PHB depolymerase and the esterase activity for the substrate was measured at 180 units, respectively (Table 3), indicating the true depolymerase nature of the enzyme.  The enzyme also showed appreciable activity with other PNP-alkanoates like PNP-acetate (PNPA) and PNP-butyrate (PNPB) which is in agreement with the  PHB depolymerase of E. minima [8] and A. fumigatus Pdf1 [7]. Absence of activity with PCL indicates that the enzyme is not a cutinase [32].Table 3
Substrate specificities ofF.  solani PHB depolymerase on different polymers.
Substrates
Activity (Units/mL/min)
PNPA
a
0.6
PNPB
b
3.2
PNPD
c
180
PCL
d
0
P(HB-Co-HV)e
180
P
aolynitrophenyl acetate; pbolynitrophenyl butyrate; pcolynitrophenyl  decanoate; pdolycaprolactone; peolymer of polyhydroxybutyrate and hydroxyvalerate.


## 3.10. Identification of PHB Hydrolysis End-Products

Identification of the aqueous end-products of PHB depolymerase reaction by paper chromatography revealedβ-hydroxybutyrate monomer as major end product of PHB hydrolysis (Figure 12). Though high-resolution analysis or HPLC/LC-MS analysis is required to confirm the above observations, this preliminary observation by paper chromatography indicated mainly monomers as the aqueous end-product of PHB hydrolysis by Penicilliumsp. S2 PHB depolymerase as in the case of Comamonas sp[33] and P. picketti[30], as against the depolymerase of A. faecalis T1,A. faecalisAE122, and P. lemoigneiwhich hydrolyze PHB mainly to the dimeric and trimeric ester of hydroxybutyrate [8, 10, 24, 27].Figure 12
Identification of PHB hydrolysis end products by PHB depolymerase ofF. solani. Lane 1—β-hydroxybutyrate standard, Lane 2—end-product of PHB depolymerase reaction.


## 4. Conclusion

Biodegradable plastics, such as PHB, continue to make progress in both the commercial and scientific fields. However, their use as a replacement for conventional plastics in a wide range of applications has been hindered by their brittleness, low mechanical strength, and high production cost. Improvements in the fermentation technology and genetically modified strains are being developed for the economical and efficient production of PHB [34, 35]. Many fine chemicals produced by chemical processes can also be prepared using microbial fermentation [36]. Our isolate, Penicillium expansum, is a good candidate for the production of pure D-3-hydroxybutyric acid, which is the main component of the degradation products obtained by enzymatic degradation of PHB [1]. A pure monomer of PHB, D-3-hydroxybutyric acid, is also an important precursor of 4-acetoxyazetidinone, which is used in making carbapenem antibiotics [37]. Intensive studies on this newfound strain as a useful microorganism for industrial application are necessary.



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*Source: 406386-2012-08-16.xml*