purification and characterization of poly(l-lactic acid)-degrading enzymes from amycolatopsis...

R E S E A R C H L E T T E R

Puri¢cationand characterizationof poly(L-lactic acid)-degradingenzymesfromAmycolatopsis orientalis ssp. orientalisFan Li, Sha Wang, Weifeng Liu & Guanjun Chen

The State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong, China

Correspondence: Weifeng Liu, State Key

Laboratory of Microbial Technology, School of

Life Science, Shandong University, Jinan

250100, Shandong, China. Tel.:186 531

88364324; fax: 186 531 88565610; e-mail:

[email protected]

Received 28 November 2007; accepted 21

January 2008.

First published online 18 March 2008.

DOI:10.1111/j.1574-6968.2008.01109.x

Editor: Alexander Steinbuchel

Keywords

Amycolatopsis orientalis ssp. orientalis ; PLA;

PLAase; serine protease.

Abstract

Polylactide or poly(L-lactic acid) (PLA) is a commercially promising material for

use as a renewable and biodegradable plastic. Three novel PLA-degrading enzymes,

named PLAase I, II and III, were purified to homogeneity from the culture

supernatant of an effective PLA-degrading bacterium, Amycolatopsis orientalis ssp.

orientalis. The molecular masses of these three PLAases as determined by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis were 24.0, 19.5 and 18.0 kDa,

with the pH optima being 9.5, 10.5 and 9.5, respectively. The optimal temperature

for the enzyme activities was 50–60 1C. All the purified enzymes could degrade

high-molecular-weight PLA film as well as casein, and the PLA-degrading activities

were strongly inhibited by serine protease inhibitors such as phenylmethylsulfonyl

fluoride and aprotinin, but were not susceptive to chymostatin and pepstatin.

Taken together, these data demonstrated that A. orientalis ssp. orientalis produces

multiple serine-like proteases to utilize extracellular polylactide as a sole carbon

source.

Introduction

Degradable polymers are increasingly considered as an

attractive alternative to the current petroleum-derived plas-

tics from the viewpoint of environmental protection and

solid-waste management. Among others, polylactide or

poly(L-lactic acid) (PLA) is a commercially promising

material for use as a renewable and biodegradable plastic,

which is readily obtained by microbial fermentation from

corn starch or renewable resources such as cane molasses

and lingo-cellulose (Gross & Kalra, 2002; Auras et al., 2004;

Tsuji, 2005).

Compared with its synthesis, relatively little has been

known for the biodegradation of PLA. Although it has been

confirmed that PLA is naturally degraded in soil or compost

and microorganisms able to degrade PLA have been de-

scribed in many reports, PLA is known to be less susceptible

to degradation in the natural environment than other

aliphatic polyesters such as poly (b-hydroxybutyrate)

(PHB) and poly (e-caprolactone) (PCL) (Pranamuda et al.,

1995, 1997). On the other hand, PLA has been considered to

be mainly degraded by proteinase-like enzymes with protei-

nase K being recognized as a typical PLA-degrading enzyme

(Williams, 1981). Furthermore, a relationship between PLA-

degrading activities and casein or silk fibroin-degrading

activities in PLA-degrading Amycolatopsis sp. and Tritira-

chium album has been reported (Jarerat & Tokiwa, 2001;

Nakamura et al., 2001). Nevertheless, an esterase as well as a

lipase have also been linked to PLA degradation (Hoshino &

Isono, 2002; Akutsu-Shigeno et al., 2003). More recently, a

PBS-degrading enzyme from Aspergillus oryzae and a cuti-

nase-like enzyme from Cryptococcus sp. strain S-2 were

reported to be able to degrade PLA (Maeda et al., 2005;

Masaki et al., 2005). Because only a few PLA-degrading

enzymes have been isolated, it is thus unclear as to how these

various enzymes with different catalytic properties achieve

the hydrolysis of PLA, and whether several enzymes are

needed simultaneously to completely degrade PLA. It is also

interesting to ask whether other more unique enzymes exist

for the efficient degradation of PLA. Moreover, considering

that enzymatic degradation is an ideal PLA waste treatment

strategy, which not only accelerates the degradation of PLA in

a controllable manner but also recycles the hydrolysate as

materials for polymer synthesis, attempts to identify and

characterize appropriate PLA-degrading enzymes seem quite

necessary (Jarerat et al., 2006; Tokiwa & Calabia, 2006).

FEMS Microbiol Lett 282 (2008) 52–58c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

In this study, we isolated a PLA-degrading actinomycete

and confirmed its ability to degrade high-molecular-weight

PLA. We also report for the first time the simultaneous

purification and primary characterization of three PLA-

degrading enzyme components from one PLA-degrading

strain.

Materials and methods

Chemicals

PLA (solid) with a molecular weight range of

85 000–160 000 was from Sigma Chemical Co. PLA powder

and film (300–500mm) with a molecular weight of 20 000

and 200 000 were purchased from Chengdu Organic Che-

micals Company Ltd of Chinese Academy of Sciences

(COCC). Poly (3-hydroxybutyrate) with an average mole-

cular weight of 270 000 was from Aldrich Co. Plysurf A210G

was from Daiichi Kogyo Seiyaku (Japan). Protease inhibitors

were from Sigma Chemical Co. Unless otherwise stated, all

chemicals used were of analytical grade.

Strain isolation, cultivation and assay of PLAdegradation

A nutrient medium containing 10 g of peptone, 10 g of

glucose, 5 g of NaCl, 2 g of yeast extract, 1.6 g of K2HPO4,

200 mg of KH2PO4 and 500 mg of MgSO4 � 7H2O in one liter

of distilled water was used for routine cultivation of the

isolated microorganisms. A mineral medium containing

0.1% (w/v) PLA or other carbon sources was used for

isolation or cultivation of PLA-degrading microorganisms

(Nishida & Tokiwa, 1993). PLA-degrading strains isolated

from soil samples and culture collections, as indicated by

growing on the PLA (MW 20 000)-emulsified agar medium

at 30 1C for 10–30 days and forming a clear zone around the

colony, were further purified with their 16S rRNA gene

sequences analyzed. Degradation of PLA by A. orientalis ssp.

orientalis was further examined by determining either the

weight loss of the PLA film (MW 200 000) or the concentra-

tion of lactate in liquid culture with sterilized PLA films as

the sole carbon source (Pranamuda et al., 1997). The surface

of PLA films after culture was observed under a scanning

electron microscope (JSM-T220) with an acceleration

voltage of 20 kV.

Purification of PLA-degrading enzymes

A 1-L culture of A. orientalis ssp. orientalis was fermented in

nutrient medium at 30 1C for 3 days and the culture super-

natant was obtained by being centrifuged at 12 000 g for

10 min. After being concentrated 10-fold by ultra-filtration

using a Pellicon XL Biomax 5 membrane (Millipore Co.),

the concentrated samples were dialyzed against 20 mM

potassium phosphate buffer (pH 7.0) and applied to a

CM Sepharose Fast Flow column (Amersham Biosciences,

Sweden) equilibrated with the same buffer. Adsorbed pro-

teins were eluted with a linear gradient of NaCl from 10 mM

to 0.5 M. Active fractions were collected and made to an

appropriate concentration of ammonium sulfate before

being loaded onto a TSK-GEL phenyl-5PW column (dia-

meter, 0.75 cm; height, 7.5 cm; Tosoh). The column was

eluted with a descending linear gradient of ammonium

sulfate from 1.3 M to 0 M. While the purified active fractions

were combined and dialyzed against 20 mM phosphate

buffer (pH 7.0), the unpurified active fractions were further

put onto a Sephadex G-50 Fine column (Amersham Bios-

ciences, Sweden), which were eluted with 0.02 M phosphate

buffer (pH 7.0) and active fractions were collected and kept

at � 80 1C. Purified enzymes were subjected to sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) and transferred onto a polyvinylidene difluoride

(PVDF) membrane, and the N-terminal amino acid se-

quences of the protein bands were analyzed using an

Applied Biosystems 491A protein sequencing system. Pur-

ified enzymes were also subjected to matrix-assisted laser

desorption/ionization time-of-flight (MALDI-TOF) mass

spectrum analysis (AXIMA-CFRplus, SHIMADZU). Selected

mass values from the MALDI-TOF experiments were taken

to search the protein nonredundant database (NR; NCBI,

Bethesda, MD) using the PeptideSearch algorithm.

Enzymatic assays

0.1% (w/v) of PLA (MW 85 000–160 000) was emulsified

with 0.01% (w/v) of Plysurf A210G and used as a substrate.

Mixtures of enzyme solution at indicated final concentra-

tions and PLA emulsion in a total volume of 2 mL were kept

at 50 1C for 6 h, except otherwise indicated. Degradation

products formed during the reaction were measured with

SBA-40C lactate biosensor (Shandong province Academy of

Sciences). One unit (U) of PLA-degrading activity was

defined as the amount of enzyme required to produce

1 mmol of lactate equivalent per minute. The caseinolytic

activity was determined with 1% (w/v) casein dissolved in

10 mM potassium phosphate buffer (pH 7.0). Briefly, pur-

ified enzymes were mixed with 1 mL casein substrate and

mixtures were incubated at 37 1C for 10 min before 3 mL of

trichloroacetic acid was added to terminate the reaction.

A275 nm was measured for the trichloroacetic acid-soluble

fraction. One unit (U) of the caseinolytic activity was

defined as the amount of enzyme required to release 1mg of

tyrosine per minute. Phenylmethanesulfonyl fluoride

(PMSF), aprotinin, chymostatin and pepstatin were used,

respectively, at their recommended concentrations that

effectively inhibit proteinases. Residual activity of purified

enzymes in the presence of these inhibitors was measured

FEMS Microbiol Lett 282 (2008) 52–58 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

53Multiple PLA-hydrolyzing enzymes isolated from Actinomycete

under standard conditions. For assay of PHB-degrading

activity, the emulsified PHB was prepared and assayed as

described by Horowitz (Horowitz & Sanders, 1994). The

esterase activity was measured by monitoring the produc-

tion of p-nitrophenyl from pNPC8 (Gao et al., 2003).

Effects of pH and temperature on the PLA-degrading activity of the purified enzymes

PLA-degrading activity of the purified enzymes was assayed

either in 50 mM phosphate buffer at pH from 5 to 8 or in

50 mM glycine–NaOH buffer at pH from 8 to 11 at 50 1C to

determine the optimal pH. The optimal temperature was

determined by measuring the PLA-degrading activity at

temperatures from 30 to 75 1C. For determination of the

pH stability and thermostability of the enzyme, purified

enzymes were kept either at pH from 4 to 11 at 4 1C for 24 h

or at indicated temperatures for different time intervals

from 0.5 h to 8 h, and residual activities were assayed under

standard conditions.

Results

Degradation of PLA by A. orientalis ssp.orientalis

Amycolatopsis orientalis ssp. orientalis was screened out from

our culture collections as the most effective PLA-degrading

microbe for its ability to form a large and clear hydrolytic

zone on the PLA-emulsified agar medium (Fig. 1a). Trans-

parent PLA film, when used as the sole carbon source,

became opaque initially with the culture and finally col-

lapsed with its surface structure being dramatically modified

(Fig. 1b and c). About 80% of PLA was degraded within

8 days according to the weight loss of the film. When the

PLA culture supernatant was incubated with the PLA-

emulsified substrate at 50 1C for 6 h, the emulsion was

clarified and acidified. In contrast, no decrease in turbidity

and pH was observed in the reaction with the culture

supernatant preheated at 100 1C for 10 min (Fig. 1d). These

results suggested that an extracellular enzyme(s) responsible

for the degradation of PLA existed.

Fig. 1. (a) Isolation of Amycolatopsis orientalis

ssp. orientalis as a PLA-degrading microorganism

for its ability to grow and form a clear zone on

PLA-emulsified agar plates. (b and c) Electron

microscope images of the surface structure

of PLA films without and with inoculation

of A. orientalis ssp. orientalis, respectively.

(d) Degradation of the PLA emulsion by the

culture supernatant of A. orientalis ssp.

orientalis. The emulsified PLA was incubated

with concentrated culture supernatant (right

tube) or with supernatant preheated at 100 1C

(left tube) at 50 1C for 6 h.


54 F. Li et al.

Purification of PLA-degrading enzymes

Although PLA powder, PLA emulsion, silk, gelatin and

peptone were all found to be effective in inducing the

production of PLA-degrading enzymes, gelatin was chosen

considering its affordability and accessibility. Three enzymes

with PLA-degrading activities were purified to homogeneity

from the culture supernatant of A. orientalis ssp. orientalis by

a combination of chromatographic steps as described in

‘Materials and methods’. Two components named PLAase II

and PLAase III were obtained after TSK-GEL phenyl-5PW

column chromatography, and another active component

named PLAase I was purified after a further Sephadex G-50

column chromatography. The purification of the enzymes

was summarized in Table 1.

The molecular masses of the three purified enzymes were

24.0, 19.5 and 18.0 kDa, respectively, as determined by SDS-

PAGE (Fig. 2). The isoelectric points (pI) of the enzymes

were all higher than 10 (results not shown). While the initial

five amino acids were the same between PLAase I (IVGGG

TAPTVSWGAQ) and PLAase II (IVGGGNATQVYSFMV)

for the determined initial 15 N-terminal amino acids, the

overall sequences were different among PLAase I, PLAase II

and PLAase III (YDVRGGDAYYINNSS). A similarity search

in the NCBI database with the obtained N-terminal se-

quences retrieved no homology target protein for PLAase I

and PLAase II. However, a similar blast revealed an 86%

identity with a serine protease from Streptomyces lividans

and a serine protease precursor from Streptomyces coelicolor

A3(2) for PLAase III. Further efforts to identify PLAase I,

PLAase II and PLAase III by mass spectrum also failed to

find the exact match in the available database, suggesting

that these enzymes may be novel proteins which have not

been registered so far. However, results of gene cloning and

sequence analysis indicated a relatively high similarity of

PLAase II and PLAase III (GenBank accession nos. EU334748

and EU362995) with serine proteases, but revealed no

homology among PLAase II, PLAase III and the published

poly(L-lactic acid) depolymerase (Matsuda et al., 2005).

Effects of pH and temperature on thedegradation of PLA by purified enzymes

The purified PLAase I, PLAase II and PLAase III exhibited

the maximum PLA-degrading activity at pH 9.5, 10.5 and

9.5, respectively, and no enzyme activity was detected under

acidic conditions (Fig. 3a). When the PLA-degrading activ-

ities of the enzymes were assayed within a temperature range

of 30–70 1C, the maximal activity of the enzymes appeared

at 60 1C for PLAase I and PLAase III, and at about 50 1C for

PLAase II (Fig. 3b). PLAase I and PLAase III were quite

stable at pH from 6 to 9, while the stability of PLAase II

decreased quickly when pH was below 7 or above 8 (Fig. 3c).

Similarly, PLAase I and PLAase III were quite stable at

temperatures up to 60 1C for 8 h, while the activity of

PLAase II decreased by 70% after 8 h at 60 1C though it

remained relatively stable up to 50 1C (Fig. 3d, e and f).

Substrate specificity of the PLA-degradingenzymes and effects of various proteinaseinhibitors

PLAase I, PLAase II and PLAase III all degraded casein

besides PLA. In contrast, they showed no activity for PHB.

Interestingly, PLAase II, but not PLAase I and PLAase III,

exhibited subtle activity for C8 ester. Comparison of the

purified enzymes with proteinase K with respect to their

PLA-degrading activity revealed that all of them demon-

strated significantly stronger PLA-degrading capabilities. In

contrast, the specific proteolytic activities of the purified

enzymes against casein were much lower than that of

proteinase K (Fig. 4). On the other hand, while both the

PLA-degrading and the caseinolytic activities of purified

enzymes were almost completely inhibited by PMSF, neither

of these activities was significantly affected by pepstatin,

Table 1. Purification of PLA-degrading enzymes from 1 L culture super-

natant of Amycolatopsis orientalis ssp. orientalis

Components

Total

proteins

(mg)

Total

activities

(U)

Specific

activities

(U mg�1)

Purification

(fold)

Yield

(%)

Dialyzed broth 163 6.52 0.04 1 100

PLAase I 12.3 2.21 0.18 4.5 34

PLAase II 4.25 0.34 0.08 2 5.2

PLAase III 1.56 0.546 0.35 8.75 8.4

Fig. 2. SDS gel electrophoretic analysis of purified enzyme fractions. M,

molecular mass marker; lane 1, concentrated culture fluid; lane 2,

PLAase I; lane 3, PLAase II; lane 4, PLAase III.



chymostatin and EDTA. Interestingly, the PLA-degrading

activity, but not the caseinolytic activity, of all three

PLAases dramatically decreased when treated with aprotinin

(Table 2). Overall, these results indicated that all the purified

enzymes are novel serine-like proteases.

Discussion

In this study, three PLA-degrading enzymes from an efficient

PLA-degrading strain, A. orientalis ssp. orientalis, were purified

and characterized. This is the first report, to our knowledge, on

the simultaneous purification of three PLA-degrading enzyme

components from one PLA-degrading strain.

Although lipases as well as polyurethane esterase have

been investigated for the degradation of low-molecular-

weight PLA, few enzymes involved in the degradation of

high-molecular-weight PLA have been identified, and de-

tailed hydrolytic mechanisms are elusive so far. A 24-kDa

PLA depolymerase (PLD), which demonstrated degrading

activities toward casein, fibrin and high-molecular-weight

PLA, has been purified from Amycolatopsis sp. strain K104-1

(Nakamura et al., 2001). The purified PLD degraded PLA in

emulsion and in solid film, ultimately forming lactic acid.

However, it should be noted that the culture supernatant of

K104-1 produced lactate oligomers as well as a lactate

monomer when incubated with PLA, implicating the ex-

istence of other unidentified enzyme factors besides PLD.

Our presented results confirmed that there existed, in the

extracellular protein pool, more than one enzyme compo-

nent with PLA-degrading activity. Although lactate was

measured as the final hydrolytic product, formation of

lactate oligomers during enzymatic degradation was not

ruled out. Efforts to identify PLAase I, PLAase II and PLAase

III by mass spectrum failed to find the exact match in the

available database. Nevertheless, results of PMF pattern

obtained by MALDI-TOF and enzymatic characteristics

0.025 (a) (b) (c)

(d) (e) (f)

0.020

0.015

0.010

0.005

0.000

0.025

0.020

0.015

0.010

0.005

0.0008 9 10 11 12 30 40 50 60 70 3

0

20

40

60

80

100

4

420

5 6 7 8 9 10 11

6 8

pH

420 6 8420 6 8

Temperature (°C)

50°C60°C70°C

50°C60°C70°C

50°C60°C

40°C

PLAaselPLAasellPLAaselll



pH

Enz

yme

activ

ity (

U)

Enz

yme

activ

ity (

U)

Rem

aini

ng a

ctiv

ity (

%)

0

20

40

60

80

100

Rem

aini

ng a

ctiv

ity (

%)

0

20

40

60

80

100

Rem

aini

ng a

ctiv

ity (

%)

0

20

40

60

80

100

Rem

aini

ng a

ctiv

ity (

%)

Time (h)Time (h)Time (h)

Fig. 3. Effects of temperature and pH on the enzymatic activity of the purified enzymes. (a and b) The dependence of enzyme activities on pH and

effects of temperature on PLAase activities. Activities were assayed under standard conditions except at different pHs and temperatures. (c) Stability of

the enzymes at different pHs. (d, e and f) Thermostability of PLAase I, PLAase II and PLAase III, respectively.

100

80

60

40

20

0PLAase l

PLAase activityprotease activity

PLAase ll PLAase lll protease K

Purified enzymes

Rel

ativ

e ac

tivity

(%

)

Fig. 4. Comparison of the purified enzymes and proteinase K with

respect to PLA hydrolytic and caseinolytic activities. Purified enzymes

(4.6mM of PLAase I, 20.5 mM of PLAase II, 3.3 mM of PLAase III) and

3.5mM of proteinase K were used to determine the PLA-degrading and

caseinolytic activities. Specific activities were expressed relative to the

maximal PLA-degrading and caseinolytic-specific activities demonstrated

by PLAase III and proteinase K, respectively. The specific PLA hydrolytic

activity of PLAase III (defined as 100%) was 0.013 U mM�1 and the

specific caseinolytic activity of proteinase K (defined as 100%) was

80 U mM�1.


56 F. Li et al.

indicated that these enzymes were probably not derived from

the same precursor. Moreover, the obtained gene sequences of

PLAase II and PLAase III revealed that the purified enzymes

were not only different from each other but also different

from the reported PLD. However, like PLD, both PLAase II

and PLAase III shared relatively high homology with serine

protease family members from actinomycete.

PLA has been considered to be mainly degraded by

protease-like enzymes, and proteinase K has been recog-

nized as a representative PLA-degrading enzyme previously.

Similar to PLD, the three purified enzymes displayed PLA-

degrading as well as caseinolytic activities despite the

absence of sequence homology. When compared with pro-

teinase K with respect to their inherent PLA-degrading and

proteolytic activities, the purified enzymes showed relatively

lower proteolytic activity against casein, but higher degrad-

ing activity against PLA than that of proteinase K. These

results suggested that, compared with proteinase K, the

PLAases may be more adaptable to catalyze the hydrolysis

of the ester bond between lactate units. Moreover, both the

PLA-degrading and caseinolytic activities of the PLAases

were inhibited by PMSF but were not significantly affected

by acid protease and chymotrypsin-type protease inhibitors,

suggesting again their close relationship with serine pro-

teases. Interestingly, aprotinin dramatically decreased the

PLA-degrading activity of all three PLAases while hardly

affecting their caseinolytic acitiviy that makes it possible to

precisely dissect the relationship between these two hydrolytic

processes at a more detailed molecular level. Taken together,

we concluded that there exist multiple serine-like enzymes

capable of completely degrading PLA from A. orientalis ssp.

orientalis, and further mechanistic characterization of these

PLAases are underway.

Acknowledgements

This work is supported by a grant from the National Natural

Science Research Program of China (No. 30570013).

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Table 2. Effects of various inhibitors on the PLA-degrading activities of the purified enzymes�

Inhibitor Concentration

Residual activity (%)

PLAase I PLAase II PLAase III

PLA degrading (%) Caseinolytic (%) PLA degrading (%) Caseinolytic (%) PLA degrading (%) Caseinolytic (%)

PMSF 1 mM 3.3 10 0 5.8 12 2.4

Aprotinin 3.7mM 19 95.6 36 98.3 33 100

Pepstatin 22mM 96 100 96 100 100 100

Chymostatin 300mM 96 94.1 95 99 98 100

EDTA 5 mM 65 98 80 99 100 100

�The purified enzyme was incubated with 0.1% (w/v) PLA emulsion or with 1% (w/v) casein in the reaction mixture containing inhibitors, as described in

‘Materials and methods’. Activities obtained without inhibitors was taken as 100%, where the PLAase activity of PLAase I was 0.18 U mg�1, PLAase II

was 0.08 U mg�1 and PLAase III was 0.35 U mg�1. For caseinolytic activities, those of PLAase I, PLAase II, and PLAase III as 100% were 276 U mg�1,

205 U mg�1 and 1450 U mg�1, respectively.



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58 F. Li et al.

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