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

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-␤-D-galactopyranoside) only below 30°C, an indication of cold-active ␤-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 ␤-galactosidase activity, belonged to glycoside hydrolase family 3, a family of ␤-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-␤-D-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-␤-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 K m (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-␤-glucosides such as helicin, MUG (4-methylumbelliferyl-␤-D-glucopyranoside), esculin, indoxyl-␤-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 ␤-glucosidases. The hydrolysis of a variety of aryl-␤-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low K m on indoxyl-␤-D-glucoside may make it useful for producing indigo.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 ␤-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, ␤-glucosidases are grouped in two glycoside hydrolase families (GHFs), 1 and 3, and the ␤-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 ␤-glucosidases possess significant ␤-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 (OR...

Identification and Characterization of Psychrotolerant Sporeformers Associated with Fluid Milk Production and Processing

“…Interestingly, a cold-active ␤-galactosidase has been identified in Paenibacillus strain C7 (81). While this enzyme may contribute to the ability of Paenibacillus to utilize lactose at low temperatures, hence facilitating growth in milk under refrigeration temperatures, it is not known whether the C7 cold-active ␤-galactosidase is conserved across Paenibacillus spp.…”

Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

“…The activities of PepF, LAP, PepQ, and BglA were measured according to methods described previously (12)(13)(14)19). To examine their thermostabilities, the proteins were preincubated in 100 mM potassium phosphate buffer (pH 7.0) for 30 min at various temperatures, and the remaining activities were measured.…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Cold active β-glucosidase have been characterized from Paenibacillus sp. Strain C7 [87], and Shewanella sp. G5 [120], and E. oxidotolerans A011 [89].…”

“…Generally fungal β-glucosidase exhibits optimal activity at pH of 4.0-6.0 [59, 69,86,92,93], nevertheless β-glucosidase with optimal pH of 2.4 has been reported from T. cylindrosporum Syzx4 [68] and that with optimal pH of 9 and 10 has been reported from Acremonium murorun LPSC 927 and Chaetomium globosum, respectively [94,95]. Bacterial β-glucosidase exhibits optimal activity at pH of 6-7 [44,87,96,97], although those with pH optima of 5, 8 and 10 has been reported from Caldicellulosiruptor saccharolyticus [98], Bacillus halodurans [99], Klebsiella pneumoniae [100], respectively. Yeast β-glucosidase reported from A. pullulans and Candida peltata exhibited optimal activity at pH 5 [80,101] and that from Kluyveromyces marxianus showed optimal activity at pH 5.5 [102].…”

“…For instance, β-glucosidase from Anoxybacillus sp. DT3-1 [83], Exiguobacterium antarcticum B7 [84], and Thermoanaerobacterium thermosaccharolyticum DSM 571 [85] has been expressed in E. coli as fusion protein with six His tag at N-terminal and purified through Nickel-Nitrilotriacetic acid (Ni-NTA) chromatography [85,86,87]. Glutathione-S-Transferase (GST) affinity has been used for purification of recombinant β-glucosidase from Exiguobacterium oxidotolerans A011 expressed as fusion protein with GST which provides additional advantage by acting as a chaperone protein to facilitate protein folding, and provide a mean for their purification by immobilized glutathione column [88,89].…”