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Applied and Environmental Microbiology, August 2005, p. 4225-4232, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4225-4232.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of an Unusual Cold-Active ß-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7

Stephanie Shipkowski* and Jean E. Brenchley

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

Received 30 November 2004/ Accepted 26 February 2005


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ABSTRACT
 
We selected for spore-forming psychrophilic bacteria able to use lactose as a carbon source and one isolate, designated Paenibacillus sp. strain C7, that was phylogenetically related to, but distinct from both Paenibacillus macquariensis and Paenibacillus antarcticus. Some Escherichia coli transformants obtained with genomic DNA from this isolate hydrolyzed X-Gal (5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside) only below 30°C, an indication of cold-active ß-galactosidase activity. Sequencing of the cloned insert revealed an open reading frame encoding a 756-amino acid protein that, rather than belonging to a family typically known for ß-galactosidase activity, belonged to glycoside hydrolase family 3, a family of ß-glucosidases. Because of this unusual placement, the recombinant enzyme (BglY) was purified and characterized. Consistent with its classification, the enzyme had seven times greater activity with the glucoside substrate ONPGlu (o-nitrophenyl-ß-D-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-ß-D-galactopyranoside). In addition, the enzyme had, with ONPGlu, a thermal optimum around 30 to 35°C, activity over a broad pH range (5.5 to 10.9), and an especially low Km (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-ß-glucosides such as helicin, MUG (4-methylumbelliferyl-ß-D-glucopyranoside), esculin, indoxyl-ß-D-glucoside (a natural indigo precursor), and salicin, but had no activity with glucosidic disaccharides or lactose. These characteristics and substrate preferences make the BglY enzyme unique among the family 3 ß-glucosidases. The hydrolysis of a variety of aryl-ß-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-ß-D-glucoside may make it useful for producing indigo.


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INTRODUCTION
 
Glycoside hydrolases cleave the bond between two carbohydrates or a carbohydrate moiety and another molecule. The classic Enzyme Classification System (EC) groups glycoside hydrolases by substrate specificity; for instance, the ß-glucosidases collectively have the designation EC 3.2.1.21. The EC system does not use structural or evolutionary information and is thus less useful for categorizing enzymes when the substrate preferences are not known. Therefore, Henrissat (18) initiated a classification system based on amino acid sequences, hydrophobicity plots, and reaction mechanisms. Under this system, ß-glucosidases are grouped in two glycoside hydrolase families (GHFs), 1 and 3, and the ß-galactosidases (EC 3.2.1.23) in four families, 1, 2, 35, and 42. Although this system illustrates enzyme relationships, it does not indicate the natural substrate or function of the enzyme. For example, some GHF 1 ß-glucosidases possess significant ß-galactosidase activity (5) and the physiological functions of many of these are unknown.

The need for biochemical and physiological studies of microbial glycoside hydrolases is highlighted by the prevalence of open reading frames (ORFs) homologous to genes encoding GHF 1 and GHF 3 enzymes in a majority of analyzed bacterial genomes (10). Although the original interest in ß-glucosidases focused on their participation in degrading the plant polymers cellulose and xylan (14), the importance of the interaction of ß-glucosidases with plant-produced compounds goes beyond catabolic degradation and includes plant-phytopathogen interactions (6, 29, 32). There is also considerable interest in the biotechnological applications of ß-glucosidases relating to plant-based foods, for instance, converting phytoestrogen glucosides in fruits and vegetables to aglycone moieties, detoxification of cassava, aroma enhancement, and removing bitter compounds from citrus fruit juices or unripe olives (5). The ß-glucosidase genes present in sequenced genomes could encode enzymes with any of these functions or, given the widespread occurrence of glucosylated compounds found in plants, additional unknown functions, some of which might contribute to plant-microbe signaling. Thus, the annotation of an ORF as a glycoside hydrolase alone provides little information about its biochemical traits or physiological function. This gap in our knowledge illustrates the need to biochemically characterize these prevalent enzymes to provide the information needed to begin understanding their biological roles.

We have been investigating psychrophilic microorganisms and their cold-active glycoside hydrolases (7, 9, 27, 37) with particular interest in their physiological roles. As part of this work, we enriched for and isolated numerous psychrophilic spore-forming bacteria to obtain phylogenetically related organisms with ß-galactosidases active on the chromogen 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside (X-Gal) at low temperatures. One transformant from a genomic library created from a Paenibacillus isolate, C7, hydrolyzed X-Gal, but sequence analysis grouped the enzyme responsible (BglY) in GHF 3, a family typically lacking appreciable ß-galactosidase activity. Because of this interesting placement of an enzyme with ß-galactosidase activity into GHF 3, we purified the enzyme and examined its thermal properties, pH dependence, substrate specificities, and kinetic characteristics.


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MATERIALS AND METHODS
 
Isolation and characterization of isolate C7.
Water and mud samples from Bear Meadows Natural Area, PA (40°44'N, 077°45'W, 554-m elevation) were inoculated into a variety of liquid minimal media with lactose as the carbon source and incubated at temperatures between 2 and 10°C for several months with periodic transfers. Spore-forming bacteria were then selected by heating aliquots at 70°C for 10 min to kill vegetative cells. The treated aliquots were inoculated into trypticase soy broth without dextrose and incubated aerobically at 2°C for 10 days. This enrichment was plated on TSA (trypticase soy agar without dextrose) modified to contain sporulation salts (36). The resulting colonies were screened for cold-active glycoside hydrolases, as indicated by the cleavage of X-Gal (100 µg/ml–1) on TSA and R2A (34) at 2 and 10°C. Isolate C7 hydrolyzed X-Gal and was studied further.

API 50 CH strips (bioMerieux, Inc. Hazelwood, MO) were used to examine the oxidation of various substrates by isolate C7. The instructions were followed except that the incubation was at 25°C and the inoculum was grown on TSA because isolate C7 did not grow on the recommended nutrient agar. The intensity of acidification was recorded at 24-h intervals for 120 h. The strips were also inoculated in combination with an M9 minimal medium (25) with phenol red (0.18 g liter–1). Growth on TSA with sodium chloride at final concentrations of 0.5, 1, 2, 3, 5, 7, and 10% was tested. Also, growth was monitored on TSA plates at 2, 10, 18, 25, 30, and 37°C.

16S rRNA gene amplification and phylogenetic analysis.
Isolate C7 cells were treated with lysozyme and the PureGene kit (Gentra Systems Inc. Minneapolis, MN) used to obtain genomic DNA with the modification of increasing the heating step to 85°C for 10 min to promote cell lysis. The 16S rRNA gene was amplified by PCR from the genomic DNA using Ready-To-Go beads (Amersham Pharmacia, Piscataway, NJ) and universal primers 8F with 907R and 704F with 1492R. Sequencing was performed at the Penn State Nucleic Acid Facility on an ABI Hitachi 3100 genetic analyzer.

The 16S rRNA gene sequence (1,458 bp) was used to search the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov) (1) (via BLAST) and the Ribosomal Database Project II (http://rdp8.cme.msu.edu/) (8). The 16S rDNA sequences of related organisms were initially aligned using Clustal W (BioEdit platform, version 5.0.6; Department of Microbiology, North Carolina State University [http://www.mbio.ncsu.edu/Bioedit/bioedit.html]), and the sequence of isolate C7 was then aligned manually. Data for the following Paenibacillus spp. were used (GenBank accession numbers follow the name of each): P. polymyxa IAM 13419T (AB042063), P. durus LMG 14658T (AJ251195), P. stellifer IS1T (AJ316013), P. odorifer TOD45T (AJ223990), P. borealis KK19T (AJ011322), P. antarcticus LMG 22078T (AJ605292), P. macquariensis DSM 2T (AB073193), P. pabuli HSCC 492T (AB045094), P. amylolyticus JCM 9906T (AB073190), P. chibensis JCM 9905T (AB073194), P. ehimensis KCTC 3748T (AY116665), P. koreensis YC300T (AF130254), P. elgii SD17T (AY090110), P. lautus JCM 9073T (AB073188), P. glucanolyticus DSM 5162T (AB073189), P. lactis MB 1871T (AY257868), and P. campinasensis JCM 11200T (AB073187).

This alignment (1,469 nucleotide positions for 18 taxa) was imported into PAUP (version 4.0b10; School of Computational Sciences and Information Technology, Florida State University [http://paup.csit.fsu.edu/]) in order to create bootstrapped (1,000-replicate) phylogenetic trees. The maximum likelihood method was performed using both the Heuristic and Fast algorithms. Distance analyses were performed using the same algorithms, in addition to the neighbor-joining algorithm (with all 10 models available in PAUP) to generate both trees and distance matrices. The Jukes-Cantor method with equal rates for variable sites was repeated with 10,000 replicates. The phylogenetic trees and distance matrixes were compared and the trees found to be congruent.

Glycoside hydrolase gene cloning.
Genomic DNA from isolate C7 was partially digested with PstI and ligated into vector p{Delta}{alpha}18 (44), a derivative of pUC18 that lacks the Escherichia coli lacZ alpha fragment. The constructs were transformed into competent E. coli ER2585F' ({Delta}lacZ lacY+) cells that were plated onto Luria-Bertani medium supplemented with ampicillin (100 µg/ml), X-Gal (100 µg/ml), and IPTG (isopropyl-ß-D-thiogalactoside; 100 µM). Plates were incubated at 37°C for 16 h and then transferred to 18°C. Three of the transformants screened positively for X-Gal hydrolysis, and plasmid preparations from these were obtained using the Promega Wizard Plus SV Miniprep kit for further subcloning.

Subcloning in p{Delta}{alpha}18 yielded a 4.7-kb PstI-EcoRI insert that conferred the ability to hydrolyze both X-Gal and X-Glc (5-bromo-4-chloro-3-indoyl-ß-D-glucopyranoside) at 18°C but not at 37°C. Sequences from this and an adjoining 0.9-kb PstI fragment were used to perform BLAST searches of the NCBI database. The sequence was examined manually and using the Signalp WWW server (3) for putative transcription and translation control regions including the –35 and –10 regions similar to the E. coli {sigma}70 promoter. The Dense Alignment Surface Method (http://www.sbc.su.se/~miklos/DAS) (11) was used to predict transmembrane regions.

Construction of an expression vector.
The PCR-Script Amp cloning kit (Stratagene) was used to create an NheI site at the beginning of the bglY gene contained in the 4.7-kb fragment. The resulting fragment and vector pET28a were each digested with SalI and NheI, ligated, and transformed into MC1061 (DE3) competent cells. The new construct, pETC7, contained the bglY gene with the coding region for a six-histidine tag at the N terminus of the protein.

Enzyme purification.
E. coli MC1061 (DE3) cells transformed with pETC7 were grown in 500 ml of terrific broth (42) with 30 µg/ml of kanamycin at 37°C until turbidity reached an optical density at 600 nm of 0.4. The culture was then moved to 18°C, IPTG added (100 µM final concentration), and incubation continued for 15 h. The cells were harvested by centrifugation (6,370 x g, 4°C, 11 min). The cell pellet was resuspended with 3 ml/g of modified Zm buffer (100 mM sodium phosphate buffer, 10 mM KCl, 1 mM MgSO4, pH 7), which does not contain ß-mercaptoethanol (25), and the cells were disrupted with a single pass through a French pressure cell (18,000 lb/in2) and centrifuged (30,996 x g, 4°C, 30 min). Saturated ammonium sulfate was added to the clarified lysate to a final concentration of 35% at 0°C and the mixture incubated for 30 min. After centrifugation (30,996 x g, 4°C, 30 min), the undialyzed supernatant was loaded onto a nickel-charged iminodiacetic acid column at 4°C.

The column wash solution was Zm buffer containing 300 mM NaCl and imidazole at three different concentrations. The column was first washed with buffer containing 5 mM imidazole, then 20 mM imidazole, and the tagged enzyme was eluted with buffer containing 150 mM imidazole. Fractions were collected, and those with the highest activity were dialyzed overnight at 4°C in 1 liter of Zm buffer and used for enzyme characterization.

Enzyme characterization.
The specific activity measurements were performed in 1.2 ml of Zm buffer with 2.2 mM ONPGlu (o-nitrophenyl-ß-D-glucopyranoside; Sigma) and incubated at 25°C for 15 min before starting the reaction with 10 µl of diluted purified enzyme. Reactions were stopped after 5 min with 0.5 ml of 0.5 M Na2CO3 and the release of o-nitrophenol immediately measured at 420 nm. One unit of activity was defined as the release of 1 µmol of o-nitrophenol per minute. Specific activity was expressed as units per milligram of protein. Protein concentrations were determined using the Bio-Rad (Hercules, California) protein assay dye reagent protocol. All assays were performed at least in triplicate. The symbols on the graphs represent the averages of the values obtained and the error bars the ranges of values.

The thermodependence of activity was assayed between 0 and 48°C for 5 min using either 2.2 mM ONPGlu or o-nitrophenyl-ß-D-galactopyranoside (ONPGal). Thermostability assays were performed by incubating aliquots of enzyme at 25, 30, 35, or 40°C. Aliquots were removed at various times and assayed with ONPGlu as described above at 25°C for 5 min.

The effect of pH on activity was determined with ONPGlu by assaying in the following buffers ranging from pH 5 to 10.9 in 0.5-pH unit increments: citric acid buffer (0.1 M, pH 5 to 6), Zm buffer (0.1 M, pH 6 to 8), Clark and Lubs pH 8.0 to 10.2 buffer (12) (0.05 M, pH 8 to 10), and sodium carbonate buffer (0.025 M, pH 10 to 10.9). The pH stability was examined by assaying with ONPGlu in Zm buffer after incubating the enzyme for 24 h in each of the previously mentioned buffers at 4°C. Activity in morpholinepropanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), and sodium phosphate buffer (all at 0.1 M) were also tested at pH 7. Activity was measured as described above.

Possible metal ion requirements were examined in two ways. First, aliquots of enzyme were dialyzed overnight at 4°C against 0.1 M MOPS (pH 7) or MOPS containing either 1 mM MgCl2, MnCl2, CaCl2, or CuCl2; 10 mM NaCl; or KCl. Second, the enzyme was treated with 50 mM EDTA for 30 min at either 0°C or 25°C, applied to a Sephadex G-25 column (Sigma), and eluted with MOPS buffer. The EDTA-treated enzyme was assayed at 25°C for 10 min in the MOPS buffer containing 2.2 mM ONPGlu with or without the above ions in both 1 mM and 10 mM concentrations, singly and in combinations. In a modification of this method, the enzyme was incubated in the 0.1 M MOPS buffer at 25°C for 30 min before initiating the reaction with ONPGlu.

Substrate preference was examined using chromogenic ONP and p-nitrophenyl (PNP) substrates at 2.2 mM at 25°C after 5 min, with one activity unit defined as the release of 1 µmol of o-nitrophenol or p-nitrophenol per minute. The substrates (Sigma) tested were ONPGlu, p-nitrophenyl-ß-D-glucopyranoside (PNPGlu), ONPGal, p-nitrophenyl-ß-D-galactopyranoside (PNPGal), o-nitrophenyl-ß-D-xylopyranoside, o-nitrophenyl-ß-D-fucopyranoside, p-nitrophenyl-ß-D-N-acetyl glucosaminide, p-nitrophenyl-{alpha}-D-glucopyranoside, and p-nitrophenyl-{alpha}-D-galactopyranoside.

The Sigma diagnostic glucose kit was used to measure enzymatic glucose release from nonchromogenic substrates. Substrates tested at 2.2 mM were the disaccharides laminaribose, cellobiose, gentiobiose, sophorose, sucrose, and lactose and the glucosides amygdalin, arbutin, salicin, helicin, puerarin, n-octyl-ß-D-glucopyranoside, indoxyl-ß-D-glucoside (indican), and esculin. Reactions subsequently used for the glucose assay were terminated by heating at 65°C for 10 min. The chromogenic substrate ONPGlu and the fluorogenic substrate MUG were also tested using this kit. Appropriate controls showed that levels of ONPGlu hydrolysis measured by glucose release were similar to those measured by the release of o-nitrophenol.

Kinetic studies.
Kinetic studies were performed with freshly purified enzyme using PNPGlu concentrations from 0.75 µM to 40 µM, ONPGlu concentrations from 2 µM to 10 µM, and ONPGal concentrations from 0.4 mM to 7 mM. The absorbance at 420 nm was monitored for 7 min at 25°C. Kinetic studies were also performed using indoxyl-ß-D-glucoside from 0.05 mM to 2 mM, monitoring the absorbance at 678 nm at 25°C for 7 min. A standard curve was produced using synthetic indigo to determine an extinction coefficient of 1.99 mM–1 cm–1 at 678 nm. The resulting data were used to determine the Km using the Enzyme Kinetics computer program (40).

Nucleotide sequence accession numbers.
The accession number for the 16S rRNA gene sequence from Paenibacillus sp. strain C7 is AY920751, and the accession number for the sequence that includes the bglY gene and surrounding open reading frames is AY923831.


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RESULTS
 
Characterization of the C7 isolate.
Numerous psychrophilic spore-forming isolates were obtained following a heat treatment to kill vegetative cells as described in Materials and Methods. The isolate designated C7 was chosen for further study because it grew on lactose and hydrolyzed X-Gal at 2°C, an indicator of cold-active ß-galactosidase activity. Microscopic characterization showed that the cells were gram-negative rods during all growth stages and that some contained ellipsoidal spores that formed terminally to subterminally in swollen sporangia. The C7 isolate developed colonies on TSA at 25 and 18°C within 48 h, at 10°C after 5 to 6 days, and at 2°C after 2 weeks, but no growth was observed at 30°C or 37°C. Colonies formed aerobically on TSA and R2A, but the isolate did not grow on nutrient agar.

Results of 16S rRNA gene phylogenetic analysis showed that isolate C7 robustly grouped within the Paenibacillus genus, a group of aerobic spore formers (2) with species that produce polymer-degrading enzymes such as xylanase (47), agarase (46), gelatinase (22), curdlanase (19), etc. Even though basal branches on some trees collapsed to polytomy, on average, the distance matrices yielded a 2.5 and 2.4% difference between isolate C7 and P. antarcticus and P. macquariensis, respectively (data not shown).

Further physiological characterization using API 50 test strips showed that, despite similarities, the phenotype of isolate C7 differed from those reported for P. antarcticus (28) and P. macquariensis (23) as well as for the related Paenibacillus spp. P. borealis (13), P. odorifer (4), and P. graminis (4) (data not shown). Some differences included the inability of isolate C7 to produce acid from glycogen, unlike P. macquariensis (13), or grow in media containing 3% NaCl, a characteristic of P. antarcticus (28). P. macquariensis has also been identified as gram negative under all conditions (23), like isolate C7, whereas P. antarcticus was observed to be gram variable (28). Based on our results, we designated the isolate C7 simply as Paenibacillus sp. C7 until future analyses determine whether it can be classified with either P. antarcticus or P. macquariensis or as a new species.

Cloning of a gene encoding ß-galactosidase activity.
Of the approximately 22,000 ampicillin-resistant transformants obtained from the genomic library from Paenibacillus sp. C7 cloned in p{Delta}{alpha}18 and expressed in E. coli ER2585F', all were white at 37°C after 16 h. However, when the plates were transferred to 18°C, three colonies became blue within 24 h, indicating X-Gal hydrolysis at the lower temperature. The clarified lysates from these transformants hydrolyzed ONPGal and ONPGlu, while clarified lysate from cells carrying the vector alone did not. The plasmids purified from these three transformants all contained 13.5-kb inserts with the same PstI restriction patterns. Following subcloning of one of these, sequencing revealed that one open reading frame, designated bglY, encoded a family 3 glycoside hydrolase.

Analysis of the bglY gene.
The gene bglY encoded the enzyme responsible for the activity on X-Gal and had 68% identity to BglB, a GHF 3 enzyme from Bacillus sp. strain GL1. Consistent with this homology, the BglY amino acid sequence possessed the conserved putative aspartate catalytic residue (21, 33) (position 246) in the motif commonly referred to as SDW but represented in this enzyme as TDW. The assignment to GHF 3 was notable because we expected the enzyme to group with families known to have ß-galactosidase activities (GHFs 2, 35, and 42) or with the one ß-glucosidase family (GHF 1) frequently found to also have ß-galactosidase activity.

The genes homologous to bglY belong to cluster F, one of several subgroups within GHF 3. This subgroup contains four enzymes as described by Cournoyer and Faure (10), two of which are known as AB' enzymes because the second domain of the enzyme, B, is "truncated" compared to AB-type GHF 3 enzymes. Our alignments (not shown) indicate that BglY is about 100 amino acids shorter than the AB enzymes, but not as truncated or condensed as the two in the AB' group, BgxA of Erwinia chrysanthemi (48) and SalB of Azospirillum irakense (15).

Analyses of bglY and neighboring sequence regions.
Further analysis was performed on bglY and adjacent sequences to determine whether an operon existed that would provide clues to the enzyme's function and relevant substrates. The sequence revealed two additional open reading frames and a partial ORF. The NCBI database comparisons of the deduced amino acid sequences assigned the COG (Clusters of Orthologous Groups) (http://www.ncbi.nlm.nih.gov/COG/) (43) classifications 1082, 0673, and 2972 to orfA, orfB, and orfC, respectively (Fig. 1). These correspond, in order, to IolE; sugar phosphate isomerases/epimerases; MviM, predicted dehydrogenases and related proteins; and predicted signal transduction proteins with C-terminal ATPase domains (Fig. 1). BglY was classified as COG 1472, BglX, ß-glucosidase-related glycosidases.



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  		FIG. 1. Diagram showing the restriction sites, putative ribosome binding sites (in bold) and putative start sites (underlined), Rho-independent transcription terminator hairpins, and orientation of the ORFs detected in the analyzed portion of the cloned insert (5.6 kb). No putative rho-independent transcription terminator or known promoter sequence was found in the 22 bp between orfA and orfB.

An examination of the fragment for potential regulatory regions showed that the sequence ANGGNGG, which resembles the "ideal" ribosome binding site in Bacillus subtilis (30), exists 10 to 11 nucleotides before a putative translation start codon for each gene (Fig. 1). The genes orfA and orfB may be expressed as an operon (Fig. 1). However, the presence of putative rho-independent transcription terminators between the other genes and no association among their homologs suggests that bglY is not cotranscribed with orfA and orfB or with orfC. Thus, these nearby genes do not currently provide clues to the physiological role of BglY.

To determine whether any of the enzymes might be membrane associated or extracellular, we analyzed the sequences using the Dense Alignment Surface Method and the Signal pWWW with neural networks trained on gram-positive data. The searches indicated that the protein encoded by orfC possesses a possible signal cleavage site and may be an integral membrane protein (as expected for a signal transduction protein). These same analyses, however, did not detect similar properties in orfA, orfB, or bglY, indicating that their putative proteins are probably intracellular and not integral membrane proteins.

Enzyme purification.
The N-terminal six-His-tagged BglY enzyme was purified for the purpose of examining its biochemical properties (Table 1). The enzyme was stable at 4°C, but the specific activity gradually decreased during storage. sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant BglY enzyme preparation showed that it was at least 95% pure and had an apparent molecular mass of 81 kDa (data not shown), which is comparable to the calculated molecular weight of 83,808 with the added His tag.


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  		TABLE 1. Purification scheme for (His-tagged) BglY from E. coli

Effects of temperature and pH on activity.
Because of its initial demonstration of ß-galactosidase activity, the BglY enzyme was first assayed with ONPGal as a substrate but was also assayed with the glucosidic substrates X-Glc and ONPGlu because of its placement in GHF 3, a ß-glucosidase assemblage. The enzyme hydrolyzed all of these substrates, and the activity with ONPGlu was seven times higher than that with ONPGal (Table 2).


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  		TABLE 2. Relative activity of the purified BglY enzyme on various chromogenic substrates as measured by ONP or PNP release at 25°C

The thermodependency of activity results showed that the highest specific activity with ONPGlu was around 30 to 35°C, whereas optimal activity with ONPGal was at 25°C (Fig. 2) and was equal to 15% of the ONPGlu activity at 25°C. These thermal optima compare well with data obtained using clarified lysate containing heterologously expressed non-His-tagged BglY (data not shown). The purified enzyme demonstrated 5% of its activity at 0°C with both substrates. Thermal stability studies using ONPGlu showed that the BglY enzyme was stable at 25°C for greater than 1 h (data not shown) but lost 23% activity after 10 min at 30°C and 85% after only 5 min at 40°C (Fig. 3).



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  		FIG. 2. Thermodependency of activity of the purified BglY enzyme with ONPGlu (-{circ}-) and ONPGal (- -{square}- -). The specific activities corresponding to 100% were 18 U/mg with ONPGlu and 3 U/mg with ONPGal.



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  		FIG. 3. Thermostability of purified BglY versus time of incubation at various temperatures: 25°C ({square}), 30°C ({diamond}), 35°C ({triangleup}), and 40°C ({circ}). The specific activity corresponding to the 100% value was 13 U/mg.

The enzyme was active over a broad pH range. The optimal activity occurred between pH 7 and 8 with Zm, with phosphate and PIPES buffers providing roughly equivalent levels of activity at pH 7 (96%, 93%). Activity in the MOPS buffer was somewhat less (80%). The enzyme retained over 75% of the optimal activity between pH values 6.5 and 9 and at least 50% between pH values 6 and 10 and had residual activity at pH 10.9 and 5.5, but none at pH 5. Inclusion of 50 mM ß-mercaptoethanol in the Zm buffer gave only 91% of the control activity. The enzyme was also stable (≥80% activity recovered after 24-h incubation in different buffers) throughout the pH range of pH 6 in Zm to 10.9 in carbonate buffer (data not shown). However, BglY lost activity in citric acid buffer at pH 6 or lower.

Effects of metal ions on activity.
The effects of metal ions on activity were first studied by dialyzing the enzyme in MOPS containing various metals and then assaying in the presence of the same metals. Compared to the activity determined in MOPS without metals, the addition of 1 mM Mg2+, 1 mM Ca2+, 1 mM Mn2+, 10 mM K+, or 10 mM Na+ had no effect. Assays with 1 mM Cu2+, however, caused a 58% loss in activity. Only a slight activity loss (19%) was observed when the enzyme was assayed at 25°C after treatment with 50 mM EDTA at 0°C for 30 min. However, this same EDTA treatment at 25°C caused a 90% loss in activity that was partially restored with the addition of the cations Ca+2, Mg+2, and Mn+2 (Table 3). A parallel control reaction demonstrated that the effects of the Sephadex column purification on the enzyme in the absence of EDTA treatment caused less than a 20% loss in activity. Incubating the treated enzyme with the metals at 25°C for 30 min prior to the assay did not affect the amount of activity recovered, nor did the addition of 10 mM KCl with 1 mM Ca+2, Mg+2, or Mn+2.


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  		TABLE 3. Effects of ions on the activity of EDTA-treated BglY enzyme

Substrate preference studies.
Synthetic chromogens were tested based on the range of substrates hydrolyzed by other GHF 3 enzymes. The chromogenic substrate yielding the highest activity was ONPGlu. The enzyme was specific for the ß-linkage (Table 2). The enzyme had greater activity on ONP substrates than PNP substrates even though studies with other GHF 3 enzymes more frequently report results using PNP chromogens. Although the enzyme possessed the greatest activity with the chromogenic ß-glucoside substrates, it also had activity with ONPGal and low activity on PNPGal and o-nitrophenyl-ß-D-fucopyranoside. Minimal activity (<1% of ONPGlu) was detected using o-nitrophenyl-ß-D-xylopyranoside and p-nitrophenyl-ß-D-N-acetyl glucosaminide.

We examined the hydrolysis of aryl substrates with different structures (Fig. 4) because the only cluster F enzyme with an identified function, SalB, is an aryl-ß-glucosidase. BglY released glucose from chromogenic, fluorogenic, and natural aryl-glucoside substrates (Table 4) but did not release significant amounts of glucose (less than 0.5% of that released from ONPGlu) from any of the disaccharides, the cyanogenic substrate amygdalin, or the synthetic substrate with an alkyl aglycone, n-octyl-ß-D-glucoside. The highest activity was with ONPGlu, followed by helicin, a partially oxidized form of salicin. Even though salicin has a similar structure, activity was less than that with helicin. Puerarin, a glucosylated flavonoid, was not significantly hydrolyzed, nor was arbutin. The enzyme was also active with MUG, a synthetic coumaric substrate, and esculin, a natural coumaric substrate. Of special interest because of the possible application for dye production was the hydrolysis of indican, an indole glucoside, with the concomitant synthesis of indigo. The blue color developed as the reaction occurred at 25°C but intensified during inactivation of the enzyme at 65°C due to the oxidation and dimerization of the intermediates to form indigo. Color formation during heating of a control with indoxyl-ß-D-glucoside but without enzyme was not significant.



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  		FIG. 4. Structures for some of the substrates used in assays with purified BglY. (A) Phenolic substrates: for ONPGlu, X{cjs0808}H, R{cjs0808}NO2; for helicin, X{cjs0808}H, R{cjs0808}CHO; for salicin, X{cjs0808}H, R{cjs0808}CH2OH; for PNPGlu, X{cjs0808}NO2, R{cjs0808}H; for arbutin, X{cjs0808}OH, R{cjs0808}H; (B) coumaric substrates: for esculin, R{cjs0808}H; for MUG, R{cjs0808}CH3; (C) indolyl substrates: for indican, X{cjs0808}Y{cjs0808}H, R{cjs0808}O-ß-D-glucose; for X-Glc, X{cjs0808}Cl, Y{cjs0808}Br, R{cjs0808}O-ß-D-glucose; for indole-3-acetic acid-glucoside, X{cjs0808}Y{cjs0808}H, R{cjs0808}CH2CO{cjs0807}O-ß-D-glucose.


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  		TABLE 4. Relative activity of the BglY enzyme on various aryl substrates as monitored by glucose release at 25°C

Kinetic studies.
The Km value for BglY was determined with PNPGlu to be low (4.9 µM) and was 2 mM with ONPGal (Table 5) at 25°C. For comparison, the Km value with PNPGlu was also determined using clarified lysate containing non-His-tagged BglY and was found to be in the same range, 3.3 ± 0.6 µM, as that measured using the tagged enzyme. The Michalis constants for ONPGlu were estimated because the substrate concentrations used at these low Km values and the ONP extinction coefficient were too low to accurately measure the initial velocity of product formation under the conditions used. However, our preliminary results indicated that the Km value for ONPGlu is also low (below 3 µM) since no significant increase in velocity occurs at greater substrate concentrations, suggesting that saturation has been attained. Kinetics were also performed with indoxyl-ß-D-glucoside by monitoring the increasing absorbance occurring at 668 nm due to indigo production (via spontaneous dimerization of the indoxyl product) at 25°C, and the Km value was determined to be 0.2 mM.


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  		TABLE 5. Kinetic values determined for purified BglY with different substrates


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DISCUSSION
 
The C7 isolate is a spore former that grows between 2 and 25°C and hydrolyzes X-Gal, consistent with the selection and screening protocols used for its isolation. The C7 isolate is clearly a member of the Paenibacillus genus; however, further characterization of the isolate may warrant its designation as a new species because it is phylogenetically at least 2.3% distant from both the P. macquariensis and P. antarcticus species. Consistent with this isolate's growth at low temperatures, the GHF 3 enzyme encoded by the cloned bglY gene is cold-active, with a thermal optimum of 30 to 35°C with ONPGlu and 5% activity at 0°C. This optimum is half the 60°C optimum of the mesophilic enzyme BglB from Clostridium thermocellum, an enzyme that has only about 5% activity at 20°C (35), with PNPGlu.

Interestingly, the thermal optimum of BglY with ONPGal is lower than that found with ONPGlu. Although infrequently studied in GHF 3 enzymes, variations in thermal optima with different substrates were observed with the GHF 3 ß-glucosidase from C. thermocellum (35). However, the divergence with that enzyme could have been due to the different assay conditions used, whereas the differences in thermal optima for BglY with ONPGlu and ONPGal were observed using identical assay conditions. As frequently found with other cold-active enzymes, BglY is heat labile and loses activity at 30°C. However, the BglY enzyme is extremely stable over a wide pH range, maintaining over 80% activity following incubation in buffers ranging from pH 6 to 10.9 for 24 h. This stability is surprising, as is the enzyme's activity over a broad pH range.

Results of initial ion studies with BglY were similar to those found with other GHF 3 ß-glucosidases with inhibition by copper, no activation by the addition of divalent ions (Mg2+, Mn2+, or Ca2+), and no effect from EDTA treatment (17, 20, 31, 45, 49). However, we also obtained results suggesting that EDTA had increased access to metal ions at a higher temperature, resulting in an enzyme that could not be restored to full activity by the reincorporation of divalent ions. This failure to restore activity may be due to an inability to obtain the proper conformation necessary for the reincorporation of metal ions that may be required for structural stability, but not catalytic activity. It is possible that this phenomenon might be observed with other GHF 3 enzymes if additional EDTA treatment conditions were used.

Not only does BglY have an unusual combination of temperature, pH, and ion characteristics, it is also atypical because it hydrolyzes both chromogenic ß-galactosides and ß-glucosides. Enzymes active on both ß-galactosides and ß-glucosides are frequently found in GHF 1, but not GHF 3. Activity with PNPGal has been reported for about 2 dozen microbial GHF 3 enzymes. With a single exception, this activity was less than 3% when compared with another appropriate substrate such as PNPGlu, PNPX, or PNPNAG In contrast, both BglB from C. thermocellum and BglY are unique in having twice this relative activity with PNPGal versus PNPGlu (35).

An additional biochemical feature of the BglY enzyme is its low Km value of 4.9 µM with PNPGlu as the substrate. This Km is 10-fold lower than that of the only cluster F enzyme with published kinetic analyses, SalB from A. irakense (50 µM, PNPGlu) (15). Enzymes in the subcluster C3, distant from cluster F (10), are the only other GHF 3 enzymes with Km values in this low range. One of these is from Thermotoga maritima with a Km of 3.9 µM on PNPGlu (16). The other is an enzyme from Agrobacterium tumefaciens with a Km value of 5 µM on PNPX (p-nitrophenyl-ß-D-xylopyranoside) (38). This enzyme also releases coniferyl alcohol from coniferin, an aryl-ß-glucosidase, as part of vir gene induction (6) and has a low Km (23 µM) on this substrate (50). The combination of the BglY enzyme's ability to hydrolyze a variety of aryl-glycosides and these low Km values makes it of biochemical interest.

In an attempt to define its physiological function, we compared BglY with other enzymes. The only other known GHF 3 enzyme from a Paenibacillus sp. (41) belongs to the distant subcluster C3, but the most homologous enzymes to BglY are those belonging to cluster F, such as BglB from Bacillus sp. GL1. (Although listed as a Bacillus sp., the 16S rDNA sequence most closely resembles sequences from other Paenibacillus spp. [data not shown]). The Bacillus sp. GL1 enzyme, BglB, has the highest identity to BglY and is well characterized, but its function is unknown. Within cluster F, SalB is the only enzyme with an identified function. This enzyme allows A. irakense to use salicin as a carbon source (15). The BglY enzyme also hydrolyzes salicin, and Paenibacillus sp. C7 grows on this compound; it will be interesting to determine whether the BglY enzyme has a function similar to SalB. However, several factors suggest that the functions of both SalB and BglY may be more complex. For one, salicin might not be the only substrate of SalB (15, 39). Second, the salB gene in A. irakense is in an operon with a second GHF 3 gene and a transporter, with a LacI-type repressor nearby, whereas bglY does not appear as part of an operon. And third, BglY lacks the signal peptide found with SalB, suggesting the two enzymes could have different cellular locations. Examination of the genes adjacent to other cluster F enzymes does not reveal any consistent gene arrangements similar to those observed with salB, or bglY, nor to each other. Thus, no conserved functional associations can be made based on operon structure.

It is possible that the physiological role of the BglY enzyme is associated with its ability to degrade substrates with aryl-aglycones because the enzyme hydrolyzes ONPGlu, PNPGlu, helicin, MUG, esculin, and salicin but not disaccharides. Therefore, the in vivo function of BglY might also involve the hydrolysis of aryl-ß-glucosidic compounds. Some possibilities could be plant metabolites such as coniferin, scopulin, mono and di-galloyl esters of glucose (precursors of tannin), indole-3-acetic acid-glucoside (Fig. 4), and glucosylated flavonoids. The combination of the enzyme's activity with several aryl-glucosides and its very low Km values suggest that it could degrade secondary plant metabolites with aryl-aglycone groups found in low concentrations. This could be environmentally relevant since Paenibacillus spp. are frequently associated with plants and their roots (4), and these possibilities will be explored.

The BglY enzyme's biochemical properties contribute to our knowledge of ß-glucosidases in general and the understudied cluster F in particular. Although putative GHF 3 enzymes are encoded in many genomes, no specific function for BglY was indicated by annotations of homologous genes. Also, there are few GHF 3 enzymes that have been assayed using a wide range of substrates for comparison. This highlights the need for the continued characterization of enzymes to supplement the experimental knowledge used for genome annotation in order to improve the precision of predicted functions. The ß-galactosidase activity observed with BglY by using chromogenic substrates is probably not relevant to lactose degradation but is a consequence of the enzyme's recognition of aryl groups, and our data suggest that BglY has a role other than the degradation of cellulose, xylan, or the cell wall components most often associated with GHF 3 enzymes. We also plan to determine whether BglY's ability to cleave indican could have applications for the production of indigo dye from Polygonum tinctorium. The specific activity of purified BglY on indican is 300 times greater than that reported for an enzyme studied for possible industrial use, Novarom G (24), and the Km of BglY with indican is slightly lower than that reported for the ß-glucosidase produced natively by P. tinctorium (26).


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ACKNOWLEDGMENTS
 
We thank A. Phillips and members of our laboratory for helpful discussions and suggestions.

This research was supported by Department of Energy grant DE-FG02-93ER20117, and Stephanie Shipkowski was partially supported by grant NSF/IGERT DGE-9972759 from the Biogeochemical Research Initiative for Education.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 211 South Frear, University Park, PA 16802. Phone: (814) 865-3330. Fax: (814) 865-3330. E-mail: sar242{at}psu.edu. Back


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Applied and Environmental Microbiology, August 2005, p. 4225-4232, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4225-4232.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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