The Protein Factory culture collection of actinomycetes belonging to different genera (Streptomyces, Actinoplanes, Nonomuraea, Microbispora and Planomonospora) was screened for the ability to degrade chitin and lignin. Then, a representative selection of species from different genera was subjected to further investigations to detect the single chitinolytic and ligninolytic activities.

Ilaria Presti1, Francesca Berini1, Loredano Pollegioni1,2, Flavia Marinelli1,2

1 Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy 2 The Protein Factory, research center of Politecnico of Milano, ICRM, CNR, Milano and University of Insubria, Varese, Italy

ilaria.presti@uninsubria.it

Metaexplore is a five years project, born from the collaboration of 18 groups all over Europe. It is driven by industrial demand and state-of-the-art environmental metagenomics technologies. Its aim is to explore the metagenomes of microbial communities of

selected soils and aquatic habitats to discover new enzimatic activities involved in the biodegradation of recalcitrant molecules, such as chitin and lignin. The most promising genes are being expressed in heterologous hosts in order to characterize the new enzymes

and exploit them for several industrial applications.

Chitin degradation

Chitosan degradation

Chitin deacetylation

Thirtythree actinomycetes were able to grow on lignin as main carbon source, but only S. coelicolor A3 formed a clear halo around the colony.

Enzyme Method/substrate

Chitin depolymerase (EC 3.2.1.14) 4-MUF β-D-N,N′,N′′-triacetylchitotrioside

Chitobiosidase (E.C. 3.2.1.52) 4-MUF β-D-N,N′-diacetylchitobioside

N-acetylglucosaminidase (E.C. 3.2.1.52) 4-MUF N-acetyl-β-D-glucosaminide

Chitin deacetylase (E.C. 3.5.1.41) Acetic acid release

All the actinomycetes tested were able to degrade chitin on agar plates. Seven actinomycetes could also degrade chitosan, as shown by Calcofluor White M2R staining of inoculated chitosan agar plates. Only three strains seemed to possess the chitin deacetylase enzyme.

The Role of Actinomycetes in the Metaexplore Project

The selected actinomycetes, grown in a liquid media containing chitin as main carbon source, had a prevalent extracellular chitin depolymerase and chitobiosidase activities, meanwhile the N-acetylglucosaminidase had an intracellular localization. After ammonium sulphate precipitation, a chitin deacetylase activity could be measured in the extracellular fraction of Nonomuraea ATCC 39727. In the presence of lignin, laccase activity was present in S. coelicolor A3 and Nonomuraea ATCC 39727: this activity was enhanced in nitrogen rich media. The manganese peroxidase activity was only detected in Nonomuraea ATCC 39727, especially in low nitrogen lignin medium. No lignin peroxidase activity could be detected in the extracellular fractions.

[1] Howard et al., J Ind Microbiol Biotechnol (2003) 30: 627-635 . [2] Kirk TK, Cullen D. Enzymology and Molecular Genetics of Wood Degradation by White-Rot Fungi in: Environmentally Friendly Technologies for the Pulp and Paper lndustry, edited by Raymond A. et al., 1998 John Wiley & Sons, Inc, pp 273-307.

Chitinases and ligninases could be exploited for several industrial, chemical and pharmaceutical applications [1][2]. Actinomycetes, for their ability in degrading these molecules, could be a valuable sources of new enzymes. The rare actinomycete Nonomuraea ATCC 29727, not only degrades chitin, but shows a chitin deacetylase activity. Regarding lignin degradation, this strain seems to possess laccase and manganese peroxidase activities. Further investigations will be devoted to this strain as cell factory for novel degrading activities.

This research activity is part of the EU FP7/2007-2011 Metaexplore project, grant agreement No. 222625.

University of Insubria is involved in the validation of methods for the functional screening of the metagenomic libraries and in the heterologous expression of chitinases in conventional (E. coli) and alternative hosts (Streptomyces spp).

Lignin degradation

Laccase (E.C. 1.10.3.2) ABTS

Lignin peroxidase (E.C. 1.11.1.14) Verathyl alchol

Manganese peroxidase (E.C. 1.11.1.13) ABTS

Suppressive soil for fungal

phytophatogens

Metagenomic library functionally and

genetically screened

E. coli BL21 (DE3)

pGEX-6P-3

18H8 GST

S. lividans TK24

pIJ86

18H8 His

His 18H8

Repression of the endogenous chitinase

activity:

YEME media

+ glucose 20 g/L

The 18 family chitinase is expressed both in E. coli and in Streptomyces spp. as heterologous hosts. The protein was purified from E. coli intracellular soluble fraction at low yields (50 µg/L). Streptomyces lividans TK24 could be used as alternative host, when its own chitinases are repressed. This can be possible in the presence of glucose, which acts as catabolite repressor. In this conditions, the 18H8 activity was detected in the extracellular fraction, when the His tag was added to the C terminus. Attempts to purify this protein are on going. Streptomyces spp. could be exploited as valuable hosts for production of secreted enzymes. In addition, for their secretory ability, they could be used as hosts for the construction of metagenomic libraries.

M 1 2 3 4 5 6 S M

70

55

40

35

25

SDS-PAGE Analysis of 18H8 purification. 1 and 2: E. coli insoluble and soluble fractions; 3:

Purification of the 18H8-GST fusion protein; 4: 18H8 after on-column cleavage from the GST-

tag; 5: Eluition with reduced glutathione of GST-tag and PreScission Protease 6: PreScission

buffer used for the on-column cleavage.

M: Molecular Weight Marker S: Standard protein, rgDAAO (36 kDa).

kDa

← 18H8-GST (72 kDa)

← 18H8 (46 kDa)

← PreScission Protease (46 kDa)

← GST (26 kDa)

Expression conditions optimization to

increase the presence of the fusion protein

in the soluble fraction.