APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2008, p. 7070–7072 0099-2240/08/$08.00⫹0 doi:10.1128/AEM.00681-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 22
Flavobacterium sp. Strain 4221 and Pedobacter sp. Strain 4236 -1,3-Glucanases That Are Active at Low Temperatures䌤† Mikkel A. Rasmussen,1 Søren M. Madsen,1 Peter Stougaard,2 and Mads G. Johnsen1* Bioneer A/S, Kogle Alle´ 2, DK-2970 Hørsholm, Denmark,1 and Department of Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark2 Received 24 March 2008/Accepted 12 September 2008
Secretion of -1,3-glucanases by the arctic bacterial isolates 4221 and 4236, related to the genera Flavobacterium and Pedobacter, was discovered. Escherichia coli and Lactococcus lactis expression of -1,3-glucanases Glc4221-1 and Glc4236-1 from the respective isolates was achieved. The enzymes hydrolyzed fungal cell walls and retained activity at low temperatures. -1,3-Glucanases relating to GH16 have been isolated from a number of organisms and expressed recombinantly in Escherichia coli (1, 7, 9). A majority of the -1,3-glucanases, if not all, have maximal activities at temperatures above 40°C. One report describes a glucanase from Fibrobacter succinogenes S85 active at cold temperatures, but this enzyme degrades only cellulose (6). In some applications, it would be advantageous to use -1,3-glucanases at lower temperatures. We collected microorganisms from cold environments and screened for -1,3glucanases using different -1,3-glucan substrates. Details of the methods and procedures used throughout this study are provided in the supplemental material. Two isolates, 4221 and 4236, were subjected to phylogenetic analysis, showing that isolate 4221 was affiliated with the genus Flavobacterium and isolate 4236 was related to the genus Pedobacter. Both isolates produced small amounts of -1,3-glucanase active at low temperatures. In order to obtain adequate protein for characterization of -1,3-glucanases from these isolates, the genes were cloned using degenerated primers and PCR amplification. Alignment of -1,3glucanases belonging to the GH16 family revealed conserved amino acid regions that were used for primer design (see Fig. S1 and Table S2 in the supplemental material). After having obtained partial gene sequences in the direct PCR approaches, the remaining information was obtained by inverse PCR. The gene from Flavobacterium sp. strain 4221, glc4221-1, encoded a -1,3glucanase (GenBank accession no. EU024301), which in BLAST and ClustalX analyses showed 89% sequence identity to a putative -1,3-glucanase from Flavobacterium johnsoniae UW101 (YP_001194781) (A. Copeland, S. Lucas, A. Lapidus, K. Barry, J. C. Detter, T. Glavina del Rio, N. Hammon, S. Israni, E. Dalin, H. Tice, D. Bruce, S. Pitluck, and P. Richardson, unpublished). A putative 16-residue-long signal sequence was predicted using the SignalP program (8), and a consensus GH16 motif was found as well as Ca2⫹ binding amino acids (5). The -1,3-glucanase gene from the Pedobacter sp. strain
4236 active at cold temperatures was cloned using an identical approach (EU024302). The deduced amino acid sequence showed a low degree of similarity to sequences retrieved from BLAST searches and included only a partial GH16 consensus motif as well as Ca2⫹ binding amino acids. The closest match was a -1,3-glucanase from Lysobacter enzymogenes N4-7 (AAN77503) (9), which displayed 54% identity. No obvious ribosome binding site that could indicate the translational start site was identified. Despite the low degree of primary similarity, the secondary and tertiary structures of the Glc4236-1 -1,3-glucanase were very similar to those of Glc4221-1, and both glucanases could be modeled over the -1,3-glucanase from Nocardiopsis sp. (see Fig. S4 in the supplemental material), using HHpred and MODELLER. Overall, the structural features of Glc4221-1 and Glc4236-1 agree with the rules of enzymes active at cold temperatures; the structure in the catalytic cavity is conserved, whereas the size of loops at the exterior of the enzymes is reduced (3, 4). Since native Flavobacterium sp. strain 4221 and Pedobacter sp. strain 4236 did not produce sufficient -1,3-glucanase, recombinant production was achieved. In E. coli, the gene glc4221-1 from Flavobacterium sp. strain 4221 was fused to the OmpA signal sequence and resulted in product secretion mainly to the growth medium (Fig. 1A). Different growth temperatures were tested, and the optimum temperature for expression of recombinant -1,3-glucanase in E. coli was found to be 30°C. Protein yield was estimated by running diluted samples on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and was determined to be 54 U/ml with a specific activity of 26 U/g. Recombinant Pedobacter sp. strain 4236 glucanase, Glc4236-1, was similarly expressed in E. coli (Fig. 2) by fusing with the OmpA signal peptide. The E. coli plasmid constructions pBMS341 and pBMS383 also resulted in product secretion to the medium, with yields of 212 U/ml. However, SDS-PAGE showed no protein bands corresponding to the Glc4236-1 product (data not shown). In contrast, E. coli isolates harboring plasmid pBMS366 did produce the Glc4236-1 protein (Fig. 1B), which could be detected only when whole cells were extracted with buffer including SDS (Fig. 1B, lane 4). This suggested that the Glc4236-1 protein from this recombinant cell construction accumulated as inclusion bodies and explains why no activity could be detected.
* Corresponding author. Mailing address: Bioneer A/S, Kogle Alle´ 2, DK-2970 Hørsholm, Denmark. Phone: 45 4516 0444. Fax: 45 4516 0455. E-mail:
[email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 19 September 2008. 7070
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FIG. 1. SDS-PAGE of extractions from recombinant strains. (A) Detection of recombinant Glc4221-1 in E. coli supernatant. Lane 1, BMS339 expressing recombinant Glc4221-1. Lane 2, negative reference BMS232. (B) Detection of recombinant Glc4236-1 in E. coli whole-cell extracts. Lanes 1 and 3, clone BMS366 expressing recombinant Glc4236-1. Lanes 2 and 4, negative control BMS232. For lanes 3 and 4, whole cells were extracted with SDS. (C) Supernatants from L. lactis strains. Lane 1, UPO762 expressing recombinant Glc4221-1 after stationary growth phase was reached. Lane 2, negative reference strain AMJ399. Corresponding migration positions of marker proteins are indicated on the left. (A) LMW-SDS marker kit (GE Healthcare). (B and C) SeeBlue Plus2 marker (Invitrogen). Arrows point to the recombinant -1,3-glucanases.
Therefore, we tested an alternative expression system based on the gram-positive bacterium Lactococcus lactis (2). Since plasmid pBMS366 was the only construct that resulted in a detectable band in SDS-PAGE, we constructed a similar plasmid for expression in L. lactis, plasmid pUPO762. Experiments with recombinant L. lactis cells harboring plasmid pUPO762 did produce active enzyme detectable by SDS-PAGE (Fig. 1C). Glc4236-1 glucanase produced in E. coli and in L. lactis showed the same activity profiles versus temperature and pH (data not shown), but unlike that of E. coli, the Lactococcus expression system was well suited for high levels of secretion, resulting in a recombinant -1,3-glucanase product with a high specific activity (approximately 40,000 U/g). Expression of Glc4221-1 in this system may well improve the specific activity to a similar degree. Recombinant -1,3-glucanases were characterized with respect to their temperature and pH optimum. Despite the different amino acid sequences, the two enzymes performed sim-
FUNGAL CELL WALL HYDROLYSIS
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FIG. 3. Plug assay. Clotrimazole contents are shown at the top. The top-row wells all include concentrated supernatant from reference strain BMS232 (Ref). The same amount of concentrated supernatant equivalent to 1,600 U of Glc4236-1 -1,3-glucanase activity was added to all wells in the bottom row. The activity in the bottom row was prepared from E. coli strain BMS341 (Enz). Incubation was for 24 h at 30°C.
ilarly. Both -1,3-glucanases showed maximal activity at pH 6 and at 30°C to 35°C and with more than 50% of maximal activity at 5°C (see Fig. S5 in the supplemental material). Analysis of substrate specificity showed that both recombinant -1,3-glucanases were able to hydrolyze a wide range of -1,3-glucan polysaccharides in addition to fungal cell walls. The hydrolysis products of the Glc4236-1 glucanase (Pedobacter sp. strain 4236) and of the Glc4221-1 glucanase (Flavobacterium sp. strain 4221) were shown to be laminaribiose (a dimer) and glucose, respectively. The inability of Glc4236-1 to degrade laminaribiose is consistent with the observation from other GH16 glucosidic enzymes, which are endohydrolases producing mainly tetramers, trimers, and dimers. Unexpectedly, the Glc4221-1 glucanase (Flavobacterium sp. strain 4221) may act in an exohydrolytic manner, producing glucose as the end product, despite similarity to endohydrolytic -1,3-glucanases. Both recombinant enzymes displayed hydrolytic activity on -1,3-glucans embedded in complex structures like fungal hyphae (see Table S3 in the supplemental material). Experiments with recombinant Glc4236-1 glucanase (Pedobacter sp. strain 4236) showed that this -1,3-glucanase could enhance the effect of sublethal concentrations of the fungicide clotrimazole (Fig. 3). Therefore, -1,3-glucanases may be useful in fungal transformations or other biotechnological applications where low-temperature cell wall disintegration is preferred.
FIG. 2. Upstream gene region of glc4236-1. The possible translation initiation sites and putative signal peptide processing sites of Glc4236-1 are shown. No obvious ribosome binding sites were identified in this region. The initial residues are shown in bold and the corresponding DNA sequence is underlined. The first underlining indicates the start of the open reading frame. Vertical arrows indicate the most likely sites of signal peptide processing. Names of corresponding recombinant plasmid constructions are shown below the amino acid sequence. In plasmids pBMS341, pBMS366, and pBMS383, the native signal peptides are substituted with OmpA. In the L. lactis construction, pUP762, the signal peptide was SP310mut2. The shown sequence segment corresponds to nucleotides 339 to 538 of the database submission.
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Bente Smith Thorup and Ulla Poulsen are thanked for excellent technical assistance. We thank the Homerule of Greenland for permission to sample microorganisms. Finally, we acknowledge Arne Skerra for providing the plasmid pMF. We thank the Danish Ministry of Science, Technology, and Innovation for partly funding this work. REFERENCES 1. Asano, T., J. Taki, M. Yamamoto, and R. Aono. 2002. Cloning and structural analysis of bglM gene coding for the fungal cell wall-lytic -1,3-glucan-hydrolase BglM of Bacillus circulans IAM1165. Biosci. Biotechnol. Biochem. 66: 1246–1255. 2. Bredmose, L., S. M. Madsen, A. Vrang, P. Ravn, M. G. Johnsen, J. Glenting, J. Arnau, and H. Israelsen. 2001. Development of a heterologous gene expression system for use in Lactococcus lactis, p. 269–275. In O.-W. Merten et al. (ed.), Recombinant protein production with prokaryotic and eukaryotic cells. A comparative view on host physiology. Kluwer Academic Press, Dordrecht, The Netherlands. 3. Feller, G. 2003. Molecular adaptations to cold in psychrophilic enzymes. Cell. Mol. Life Sci. 60:648–662.
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