Substrate Specificities of Bacterial Polyhydroxyalkanoate ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1995, p. 3113–3118 0099-2240/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 61, No. 8

Substrate Specificities of Bacterial Polyhydroxyalkanoate Depolymerases and Lipases: Bacterial Lipases Hydrolyze Poly(v-Hydroxyalkanoates) ¨ CHEL,2 KARL-ERICH JAEGER,1 ALEXANDER STEINBU

AND

DIETER JENDROSSEK3*

¨r Lehrstuhl fu ¨r Biologie der Mikroorganismen der Ruhr-Universita ¨t Bochum, 44780 Bochum,1 Institut fu Mikrobiologie der Westfa ¨lischen Wilhelms-Universita ¨t Mu ¨nster, 48149 Mu ¨nster,2 and Institut fu ¨r Mikrobiologie der Georg-August-Universita ¨t Go ¨ttingen, 37077 Go ¨ttingen,3 Germany Received 5 April 1995/Accepted 17 May 1995

The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an v-hydroxyalkanoic acid such as poly(6-hydroxyhexanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly(3-hydroxybutyrate) and other poly(3hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested.

Polyhydroxyalkanoates (PHA) are biodegradable polyesters which are synthesized and accumulated intracellularly during unbalanced growth by a large variety of bacteria. They are deposited in the form of inclusion bodies and can amount to more than 90% of the dry cell mass (13, 38). Besides poly(3-hydroxybutyrate) [P(3HB)], other short-chain-length (PHASCL; 3 to 5 carbon atoms per monomer) or medium-chain-length (PHAMCL; 6 to 14 carbon atoms) PHA have been detected (reviewed in references 1, 11, 33, 40, 41, 43). The monomeric composition of PHA depends on the bacterial species as well as the carbon sources supplied (10, 14, 47). Because of their thermoplastic properties and their synthesis from renewable resources, PHA are of biotechnological interest, and P(3HB) and its copolymers with 3-hydroxyvalerate have been commercialized as BIOPOL. The ability to degrade extracellular PHA depends on the secretion of specific PHA depolymerases which hydrolyze the polymer to water-soluble products (7, 8) and is widely distributed among bacteria (5, 29). Aerobic and anaerobic PHAdegrading bacteria of many taxa were isolated from various ecosystems, and several PHA depolymerases were isolated and characterized (12, 20, 37, and references cited therein). All purified depolymerases were specific for P(3HB) and/or other PHASCL, such as the P(3HB) depolymerases of Alcaligenes faecalis, Comamonas sp., or Pseudomonas lemoignei (20, 32, 35), or for PHAMCL, such as the poly(3-hydroxyoctanoate) [P(3HO)] depolymerase of Pseudomonas fluorescens GK13 (37). Whereas most PHA-degrading bacteria analyzed so far apparently produced only one PHA depolymerase, P. lemoignei was found to have at least five PHA depolymerases (6, and references cited therein). The structural genes of several PHA depolymerases have been cloned and sequenced in the

last 5 years (18, 19, 21, 24, 35, 36). Biochemical analysis of the purified depolymerase proteins and analysis of the DNA-deduced amino acid sequences revealed that PHA depolymerases apparently possess a catalytic triad consisting of serine, histidine, and aspartate residues which have been demonstrated to form the active site in bacterial lipases (see reference 17 for a recent review). In addition, the hydrolysis of several PHASCL by lipases has been reported recently (31). These two pieces of information together with the commercial potential of biodegradable polymers prompted us to study the substrate specificities of various PHA depolymerases and lipases in detail. We analyzed the abilities of several bacterial PHA depolymerases, an esterase of P. fluorescens GK13, and five bacterial lipases to hydrolyze various PHASCL, PHAMCL, and triolein. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are given in Table 1. Source of purified PHA depolymerases and purified lipases. The previously purified extracellular P(3HB) depolymerases A and B and the poly(3-hydroxyvaleric acid) [P(3HV)] depolymerase of P. lemoignei (32), the P(3HB) depolymerase of Comamonas sp. (20), and the P(3HO) depolymerase of P. fluorescens GK13, as well as a partially purified esterase of the same bacterium, were used (37). All depolymerase proteins (Table 1) had been stored at 2208C, and their activity was confirmed before use. Extracellular lipases were purified from bacterial culture supernatants, and samples were kindly provided by Onno Misset, Gist-brocades, Delft, The Netherlands (P. alcaligenes); Shamkant Patkar, Novo Nordisk, Bagsværd, Denmark (Burkholderia cepacia); and Charles Colson, Universite´ Catholique de Louvain-La Neuve, Louvain-La Neuve, Belgium (Bacillus subtilis). P. fluorescens lipase isolated from an overexpressing Escherichia coli strain was kindly provided by Joon S. Rhee, Korea Advanced Institute of Science and Technology. The P. aeruginosa lipase was purified as described previously (30). Gel electrophoresis. Proteins were separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12% gels (21). Mr standards (from Sigma, Deisenhofen, Germany) were bovine albumin (67,000), egg albumin (43,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (30,000), trypsinogen (24,000), trypsin inhibitor (20,100), and a-lactalbumin (14,200). Gels were stained with silver (3).

* Corresponding author. Mailing address: Institut fu ¨r Mikrobiologie der Georg-August-Universita¨t Go ¨ttingen, Grisebachstrasse 8, 37077 Go ¨ttingen, Germany. Phone: 49-551-39-3777. Fax: 49-551-39-3793. 3113

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APPL. ENVIRON. MICROBIOL. TABLE 1. Bacterial strains, plasmids, and proteins Relevant characteristic

Strain

Abbreviation Pheno- or genotype

Hydrolase

Purified protein (mg/ml)

Source or reference

Bacillus subtilis Pseudomonas alcaligenes

Triolein1 Triolein1

Lipase Lipase

LipBsu LipPal

0.65 0.50

Pseudomonas aeruginosa Pseudomonas fluorescens Burkholderia cepacia

Triolein1 Triolein1 Triolein1

Lipase Lipase Lipase

LipPae LipPfl LipBhce

0.20 0.025 2.0

Pseudomonas lemoignei

P(3HB)1 P(3HV)1 P(3HB) depolymerase B P(3HV) depolymerase P(3HB) depolymerase A P(3HB) depolymerase P(3HO) depolymerase EsterasePfl

PhaZ2Ple PhaZ4Ple PhaZ5Ple PhaZCsp PhaZPfl

0.72 0.41 0.75 0.090 0.15 0.30

BCL1051 (27) O. Misset (see Materials and Methods) 45 26 S. Patkar (see Materials and Methods) LMG2207 (8) 32 32 32 DSM6781 (20) DSM7139 (37) 37

pUC92::phaZ1 pUC91::phaZ2

P(3HB) depolymerase C P(3HB) depolymerase B

PhaZ1Ple-reca PhaZ2Ple-rec

0.088 0.13

19 19

pBluescript::phaZ4 pBluescript::phaZ5

P(3HV) depolymerase P(3HB) depolymerase A

PhaZ4Ple-rec PhaZ5Ple-rec

0.63 0.032

19 19

Comamonas sp. Pseudomonas fluorescens GK13 Escherichia coli JM83 pSN654 pSN625 Escherichia coli XL1blue pSN874 pSN885 a

P(3HB)1 P(3HO)1 esterase1

Ple-rec, protein of P. lemoignei purified from recombinant E. coli.

Production of PHA. The homopolyesters P(3HB) and P(3HV) were produced by cultivating, respectively, Alcaligenes eutrophus H16 (ATCC 17699) or Chromobacterium violaceum (DSM 30191) aerobically in a 300-liter steel fermentor at 308C. The mineral salts medium described by Schlegel et al. (39), with sodium gluconate (23; 2%, wt/vol) for A. eutrophus or sodium valerate (53; 0.2%, wt/vol) for C. violaceum, was used for growth (42). Poly(4-hydroxybutyric acid) [P(4HB)] homopolyester was produced by cultivating the P(3HB) leaky mutant A. eutrophus JMP222-PHB2102, which harbored plasmid pVK101::PP1, aerobically in a 10-liter glass fermentor at 308C with 4-hydroxybutyric acid (23; 0.5%, wt/vol) as sole carbon source according to Steinbu ¨chel et al. (44). A copolyester consisting of almost equimolar amounts of 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HHX) plus 5 mol% 3-hydroxyoctanoic acid (3HO) [P(3HB-co-3HHX-co-3HO)] was produced by cultivating Pseudomonas putida GPp104 harboring plasmid pHP1014::E156 aerobically in a 10-liter glass fermentor at 308C (28). A copolymer consisting of mainly 3HO with minor amounts of 3HHX [P(3HO)] and a copolymer consisting of 3HO and 3-hydroxydecanoic acid (3HD) as main components with 3HHX and 3-hydroxydodecanoic acid (3HDD) as minor components [P(3HO-co-HD)] were produced by Pseudomonas oleovorans (ATCC 29347) and P. putida KT2440 (43) grown in defined medium (39) with sodium octanoate (0.50% 1 0.25%, wt/vol) or sodium gluconate (1.5%, wt/vol), respectively. BIONOLLE (product 3010; a polymer of aliphatic dicarbonic acids and aliphatic diols), polycaprolactone [PCL; poly(6-hydroxyhexanoate); product P-767], and polylactid were gifts from Showa Denko (Europe) GmbH, Du ¨sseldorf, Germany; Union Carbide, Bound Brook, N.J.; and Boehringer Ingelheim, Ingelheim, Germany, respectively. Isolation and analysis of PHA. PHASCL were isolated by digestion of the bacteria with sodium hypochlorite and by subsequent purification of the granules by extraction with acetone-diethyl ether (2/1, vol/vol), as described previously (20, 32). PHAMCL were isolated from lyophilized cells by solvent extraction and precipitation from the chloroform solution in the presence of an excess of ethanol, as described recently (28). Quantitative determination of PHA was done by gas chromatographic analysis of the methyl esters, which were obtained by sulfuric acid-catalyzed methanolysis according to Brandl et al. (4) and Timm et al. (46). P(3HO) contained about 92 mol% 3HO and 8 mol% 3HHX. P(HO-co-HD) consisted of 3 mol% 3HHX, 20 mol% 3HO, 72 mol% 3HD, and 5 mol% 3HDD. The copolyester consisting of 3HB plus medium-chain-length 3-hydroxyalkanoic acids contained 44 mol% 3HB, 51 mol% 3HHX, and 5 mol% 3HO [P(3HB-co-3HHX-co-3HO)]. Preparation of polymer suspension. Suspensions of P(3HB) and P(3HV) granules were prepared as described previously (20, 32). The method of preparing PHAMCL suspensions was applied according to Marchessault (28a). PHAMCL was dissolved in acetone at a final concentration of 2 g/liter. Four volumes of PHAMCL-acetone solution was added slowly with stirring into 1 volume of cool (5 to 108C), distilled water. Then the organic solvent was removed by evaporation, and a milky PHAMCL suspension in water ('8 g/liter) was obtained. Sus-

pensions of PCL were prepared by the same procedure. However, it was necessary to heat all solutions to 50 to 608C. Solution-cast films of P(4HB), P(3HBco-3HHX-co-3HO), and BIONOLLE were prepared from 2.5% (wt/vol) solutions of the polyesters in chloroform to which traces of Sudan red had been added (37). Polylactid was suspended in water (3%, wt/vol) and sonicated like PHASCL before use. Assay for PHA depolymerase activity. A quick and simple procedure for estimating the activities of P(3HB) and P(3HV) depolymerases was performed by a spot test on indicator plates: 15 ml of hot liquid medium containing 0.2 to 0.6% (wt/vol) sonicated polymer granules or polymer suspension and 1.4% (wt/vol) agar in 100 mM Tris-HCl (pH 8.0) was poured into petri dishes. After solidification, 1 to 3 ml of enzyme solution was dropped onto the surface, and the dishes were incubated at 378C for several hours. The diameters of the resulting clearing zones semiquantitatively correlated with the activities of the depolymerases (21). For estimation of P(4HB)-, P(3HB-co-3HHX-co-3HO)-, and BIONOLLE-specific depolymerase activity, 1 to 3 ml of enzyme solution was dropped onto the surface of a solution-cast film of the polymer (3 ml of a 2.5% [wt/vol] solution in chloroform), which was layered onto 20 ml of 1.4% (wt/vol) agar in 100 mM Tris-HCl (pH 8.0) in petri dishes. A quantitative assay of the enzymatic hydrolysis of PCL was performed by measuring the initial decrease of the optical density (OD) of diluted PCL suspensions at 650 nm in 1-ml cuvettes at 378C. The assay mixture contained 100 ml of PCL suspension ('0.8%, wt/vol), 5 to 50 ml of enzyme solution, and 100 mM Tris-HCl to a total volume of 1 ml. The apparent extinction coefficient, ε, of the PCL solution was determined by measuring the OD650 of various concentrations of PCL solution in 100 mM Tris-HCl and amounted to 0.00159 ml 3 mg21 3 cm21. All subsequent experiments were performed with the same PCL suspension. The hydrolysis of 1 mg of PCL per min was defined as 1 U. For Burkholderia cepacia lipase, reaction buffer contained 20% (vol/vol) ethanol. Quantitative assays for polyhydroxybutyrate depolymerase and polyhydroxyvalerate depolymerase activities were performed as described previously (20, 21). Assays for lipase activity. Activity of lipases was assayed spectrophotometrically with p-nitrophenylpalmitate (48) or 1,2-dilauryl-rac-glycero-3-glutaric acid resorufinester (data sheet to product no. 1179 934; Boehringer, Mannheim, Germany) as substrates. Qualitative determination of lipase activity was done with a rhodamine B-triolein plate assay (23). Purification and identification of hydrolysis products. After enzymatic polymer hydrolysis, the protein was precipitated with trichloroacetic acid (235 mM) and removed by centrifugation. The hydrolysis products (supernatant) were loaded on a Chromabond C18ec column (500-mg sorbent weight, 3-ml bed volume equilibrated with 2 bed volumes of methanol and 2 bed volumes of water; Macherey-Nagel, Du ¨ren, Germany), washed with 3 bed volumes of water, and finally eluted with 2 ml of chloroform. The products were concentrated by evaporation of the solvent and subsequently analyzed by direct chemical ionization in the presence of ammonia followed by mass spectrometry (nuclear magnetic resonance [2]) (model 95 spectrometer; Finnigan MAT, Bremen, Germany) at 200 eV.

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SUBSTRATE SPECIFICITY OF PHA DEPOLYMERASES AND LIPASES

RESULTS Biochemical characterization of lipases and PHA depolymerases. The lipases we used originated from the gram-positive bacterium B. subtilis and from the three groups of Pseudomonas lipases, namely, P. aeruginosa and P. alcaligenes of group I, Burkholderia cepacia (formerly P. cepacia [15]) of group II, and P. fluorescens of group III (Tables 1 and 2) (17). The determination of their specific activities revealed that Pseudomonas group I lipases of P. alcaligenes and, in particular, P. aeruginosa showed the highest activities with both p-nitrophenylpalmitate and 1,2-dilauryl-rac-glycero-3-glutaric acid resorufinester as substrates (Fig. 1A). However, all lipases tested were able to hydrolyze both substrates, thereby confirming that they belonged to the group of so-called true lipases (EC 3.1.1.3). SDS-PAGE analysis identified the lipases with respect to their Mrs (19,400 for B. subtilis; 33,000 for Burkholderia cepacia; 30,000 for P. alcaligenes; 29,000 for P. aeruginosa; and 48,000 for P. fluorescens) and revealed that all samples were of high purity (Fig. 1B). Four PHA depolymerases specific for PHASCL, i.e., P(3HB) depolymerase A (PhaZ5Ple), P(3HB) depolymerase B (PhaZ2Ple), P(3HV) depolymerase (PhaZ4Ple) of P. lemoignei, and P(3HB) depolymerase of Comamonas sp. (PhaZCsp); one PHA depolymerase specific for PHAMCL, i.e., P(3HO) depolymerase of P. fluorescens GK13; and an esterase of the same bacterium were used in this study (Tables 1 and 2). In addition, four PHA depolymerases of P. lemoignei purified from recombinant E. coli, i.e., P(3HB) depolymerase C (PhaZ1Ple-rec), P(3HB) depolymerase B (PhaZ2 Ple-rec ), P(3HV) depolymerase (PhaZ4Ple-rec), and P(3HB) depolymerase A (PhaZ4Ple-rec), were also used. In contrast to the corresponding wild-type enzymes purified from P. lemoignei, the proteins purified from recombinant E. coli and all other enzymes were not glycosylated (6, 20). Details of the purification and biochemical characterization of the PHA depolymerases were described earlier (20, 21, 32, 37). Determination of substrate specificities of purified PHA depolymerases and lipases. The substrate specificities of nine PHA depolymerases (PhaZ), one esterase, and five lipases were investigated (Table 1). Besides the homopolyesters P(3HB), P(4HB), P(3HV), PCL, and polylactid, the copolyesters P(3HHX-co-3HO), P(3HO-co-3HD), P(3HB-co-3HHXco-3HO), and BIONOLLE were used as substrates. The results are shown in Table 2. Polylactid was not hydrolyzed by any of the enzymes, thus indicating that polyesters of a-hydroxyalkanoic acids were apparently not suitable substrates for bacterial lipases and PHA depolymerases. All P(3HB) depolymerases hydrolyzed P(3HB), and PhaZ1Ple and PhaZ4Ple of P. lemoignei also hydrolyzed other PHASCL such as P(4HB) and/or P(3HV). No differences regarding substrate specificity were found for the P. lemoignei PHA depolymerases whether they were isolated from an endogenous source or from recombinant E. coli. Under the conditions applied, no PHASCL depolymerase was able to hydrolyze P(3HB-co-3HHX-co-3HO), although the polymer contained 44 mol% 3HB. Interestingly, P(3HB-co-3HHX-co-3HO) was hydrolyzed by the P(3HO) depolymerase of P. fluorescens in addition to P(3HO). PHASCL were not hydrolyzed by the P(3HO) depolymerase. In addition, none of the PHASCL depolymerases investigated in this study hydrolyzed PHAMCL or triolein. All lipases and the esterase of P. fluorescens GK13 hydrolyzed triglycerides but not PHASCL. This indicated that the esterase is also a true lipase. PHAMCL consisting of hydroxyalkanoates with the hydroxyl group in the b-position were partially hydrolyzed by the lipases of P. aeruginosa and P.

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alcaligenes as indicated by partial clearing of the opaque test medium after a prolonged incubation. However, most lipases, except that of P. fluorescens, formed complete clearing zones with BIONOLLE and PHA consisting of an v-hydroxyalkanoate such as 6-hydroxyhexanoate or 4-hydroxybutyrate. This indicated that these lipases hydrolyzed the polymer completely to water-soluble products (Table 2). These results demonstrated that small differences in the polymer structure, such as the presence or absence of side chains in the polymer backbone, highly influenced the biodegradability of the polymer. Hydrolysis of PCL by lipases. Because of the relatively high enzyme activity in the clearing zone test, the hydrolysis of PCL by lipases was studied in more detail. For quantification of the rate of hydrolysis, the initial rate of decrease of turbidity of a PCL suspension by the enzymes was measured. Because of the high turbidity of the PCL suspension, the concentration of the polymeric substrate in the assay mixture had to be relatively low (0.8 mg of polymer per ml) in order to make measurements in the range of turbidities in which the turbidity is linearly proportional to the polymer concentration. Therefore, it was necessary to determine the specific enzyme activity at a relatively low substrate concentration. When the dependence of the hydrolysis rate on the amount of P. aeruginosa lipase at a constant polymer concentration was determined, the graph was linear only up to 30 ng of enzyme per ml of assay mixture (Fig. 2). With a protein concentration of less than 30 ng/ml, a specific PCL depolymerase activity of 1.8 3 106 U/mg was calculated for the P. aeruginosa lipase. Under similar conditions, specific PCL depolymerase activities of 6,000, 40,000, and 140,000 U/mg were calculated for the lipases from B. subtilis, Burkholderia cepacia, and P. alcaligenes, respectively. Identification of the products of PCL hydrolysis by P. aeruginosa lipase. In order to identify the hydrolysis products and to elucidate whether the decrease of the OD in the activity tests was due to a cleavage of the ester bonds or to another physical process, such as emulsification of the polymer, the molecular masses of the reaction products were determined by direct chemical ionization (NH3)-nuclear magnetic resonance. Twenty milligrams of PCL (2.5 ml of the PCL suspension [0.8%, wt/vol, in water] plus 0.5 ml of 1 M K2HPO4) was hydrolyzed in the presence of 1.2 mg of purified P. aeruginosa lipase at 378C. After 2 h of incubation, the PCL suspension had become clear. In order to ensure complete hydrolysis, an additional 0.6 mg of lipase was added, and the reaction mixture was incubated for 2 more hours. The products were then purified by precipitation of the protein by trichloroacetic acid and by hydrophobic interaction chromatography. Eight milligrams of product was recovered and analyzed by direct chemical ionization-(NNH3)-nuclear magnetic resonance. A main signal at Mr 246 and minor signals at Mr 132 and Mr 202 were obtained. The main signal at Mr 246 corresponded to the molecular mass calculated for dimeric hydroxyhexanoic acid and one of the minor signals at Mr 132 corresponded to monomeric hydroxyhexanoic acid. However, when PCL (2.5 g) was extracted with ethanol (10 ml), minor signals at Mr 132 and at Mr 202 appeared also. Obviously, these compounds are unpolymerized impurities of the polymer. We concluded that the decrease of the OD of the PCL suspension was due to the lipase-catalyzed hydrolysis of PCL and that the dimer was the main hydrolysis product. DISCUSSION Several different bacterial lipases have been characterized biochemically, and the corresponding genes have been cloned and sequenced (17). The structures of lipases, which have been

2 1 1 (1) 1 2

2 2 2 2 2 2

2 2 6 2 2 1 2 2 2

18,000 16,000 32,000 11,000 14,000 23,000 22,000 16,000

P(4HB) reaction

2

1111 1111 1111 111 111 111 11 11

Sp act Reaction (U/mg)

P(3HB)

2 2 2 2 2 2

2

2 2 11 1 2 11 2 2 250

430 890 9,300 3,000

Sp act Reaction (U/mg)

P(3HV)

2 (1) NDe 2 2 2

1

2 2 2 2 2 2 2 2

P(44 mol% 3HBco-51 mol% 3HHXco-5 mol% 3HO) reaction

((1)) 111 (1) 11 1 2

((1))

2 2 2 2 2 2 2 2

Reaction

1.8 3 106 6,000 40,000 140,000 ND

Sp act (U/mg)

PCL

6 ((1)) 2 2 ((1)) 2

11

2 2 2 2 2 2 2 2

P(8 mol% 3HHXco-92 mol% 3HO) reaction

2 ((1)) 2 2 ((1)) 2

(111)

2 2 2 2 2 2 2 2

P(3 mol% 3HHX-co20 mo% 3HO-co-72 mol% 3HD-co-5 mol% 3HDD) reaction

2 11 ND 111 1 2

2

1 2 11 (1) 2 111 2 (1)

2 2 2 2 2 2

2

2 2 2 2 2 2 2 2

BIONOLLE Polylactid reaction reaction

1 111 111 111 111 111

2

2 2 2 2 2 2 2 2

Reaction on rhodamine plates

a 2, no clearing zone; 6, reaction not unequivocal; (( )), only very small and incomplete clearing zone; ( ), incomplete clearing zone; 1, small clearing zone (diameter, 4 to 6 mm); 11, medium clearing zone (diameter, 7 to 10 mm); 111, large clearing zone (diameter, 10 to 15 mm); 1111, very large clearing zone (diameter, .15 mm). b In some cases the specific activities were measured photometrically, and the values indicate the specific activity in micrograms of hydrolyzed polymer per minute and per milligram (U/mg). c For complete monomeric compositions of the polymers, see Materials and Methods. d rec, protein purified from recombinant E. coli. e ND, not determined.

PHASCL depolymerase PhaZ5Ple PhaZ2Ple PhaZ4Ple PhaZ1Ple-recd PhaZ2Ple-rec PhaZ4Ple-rec PhaZ5Ple-rec PhaZCsp PHAMCL depolymerase PhaZPfl Lipase EsterasePfl LipPae LipBsu LipBhce LipPal LipPfl

Protein

Polymerc

TABLE 2. Substrate specificitiesa and specific activitiesb of purified PHA depolymerases and lipases

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SUBSTRATE SPECIFICITY OF PHA DEPOLYMERASES AND LIPASES

FIG. 1. Biochemical characterization of lipases. (A) Specific lipase activities were determined with p-nitrophenylpalmitate (p-NPP; OD410) and 1,2-dilaurylrac-glycero-3-glutaric acid resorufinester (DGGR; (OD535) as substrates. (B) SDS-PAGE analysis of lipases obtained from bacterial strains indicated in panel A. Gel was stained with silver; lane M shows Mr marker proteins as described in Materials and Methods.

determined by X-ray crystallography, revealed a conserved folding pattern which has been referred to as the a/b-hydrolase fold and recognized as a ‘‘consensus’’ fold found in different hydrolases (34). The main structural feature in these hydrolases is a central part consisting of predominantly parallel b-sheets with the active site located inside the protein (reviewed in reference 9). The central active-site serine residue in lipases is part of a consensus pentapeptide, Gly-X1-Ser-X2-Gly, which is conserved in nearly all lipases. This central serine residue acts as a nucleophil which is stabilized by a histidine and an aspartate (or glutamate) residue, with all three residues forming the catalytic triad. A tetrahedral transition state inter-

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mediate is formed and is stabilized by hydrogen bonding with residues of the so-called oxyanion hole. The exact stereochemistry of the oxyanion hole is not yet known; however, a conserved sequence motif, His (Arg)-Gly-X-X-Gly, has been described as residing at the N-terminal part of mature lipase proteins (16) and is presumably involved in the formation of the oxyanion hole. Recently, the primary structures of eight bacterial PHA depolymerases have been analyzed (18). Although no significant overall sequence homology to lipases or to other hydrolases was found, the lipase consensus sequence motif Gly-X1-SerX2-Gly was observed in all PHA depolymerases. Interestingly, the X1 residue was a leucine in PHA depolymerases instead of a histidine in bacterial lipases. In addition, another conserved region (His-Gly-Cys-X-Gln) which seems to resemble the oxyanion hole consensus sequence in lipases was found. These findings suggested that lipases and PHA depolymerases may share a comparable mechanism of substrate hydrolysis and prompted us to study the substrate specificities of both classes of hydrolases. None of the PHA depolymerases tested showed significant lipase activity. This result demonstrated that the depolymerases were unable to (i) bind a long-chain triacylglycerol or (ii) hydrolyze this lipase substrate. However, four of the five lipases tested were able to hydrolyze polyesters of v-hydroxyalkanoic acids such as PCL, P(4HB), and BIONOLLE. A comparable observation has been described for several lipases of mainly eukaryotic origin (31). Furthermore, we determined an exceptionally high specific activity of P. aeruginosa lipase towards PCL as a substrate (1.8 3 106 U/mg), and the dimeric ester of hydroxyhexanoic acid was detected as a main hydrolysis product. In similar experiments with Pseudomonas lipase, oligomers have been identified as hydrolysis products (22). We assume that other polyesters of v-hydroxyalkanoic acids may also be suitable substrates for bacterial lipases. A weak but clearly detectable hydrolysis of PHAMCL such as P(3HO) was observed only with the lipases from P. alcaligenes and P. aeruginosa. This indicated that the presence of alkyl side chains in a polyester inhibits or at least drastically reduces its suitability as a lipase substrate. Our finding that bacterial lipases possess the ability to degrade PHA is interesting for two reasons. (i) It provides further evidence of the remarkable versatility of lipases with respect to hydrolysis of different substrates. (ii) The increasing commercial potential of various PHA, which is mainly based on their biodegradability, may be further substantiated by the fact that not only specific PHA depolymerases but also lipases produced by abundant bacteria can contribute to the biodegradation of these polymers.

ACKNOWLEDGMENTS

FIG. 2. Dependence of PCL hydrolysis rate on lipase concentration. The rates of PCL hydrolysis were calculated from the initial decrease of the OD650 of a PCL suspension by various amounts of purified P. aeruginosa lipase.

We thank Astrid Behrends and Andreas Schirmer (Universita¨t Go ¨ttingen), Onno Misset (Gist-brocades, Delft, The Netherlands), Shamkant Patkar (Novo Nordisk, Bagsværd, Denmark), Charles Colson (Universite´ Catholique de Louvain-La Neuve, Louvain-La Neuve, Belgium), and Joon S. Rhee (Korea Advanced Institute of Science and Technology, Seoul, Korea) for providing samples of purified bacterial PHA depolymerases and lipases. The performance of nuclear magnetic resonance studies by G. Remberg is also gratefully acknowledged. Additionally, we thank Boehringer Ingelheim, Showa Denko (Europe) GmbH, and Union Carbide for providing polylactid, BIONOLLE, and PCL, respectively. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Je-152 2/2) and the BRIDGE T-project on lipases (BIOT CT910272).

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