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

• Published in: Applied and Environmental Microbiology
• Main Authors: Shipkowski, Stephanie, Brenchley, Jean E.
• Format: Article in Journal/Newspaper
• Language: English
• Published: American Society for Microbiology 2005
• Subjects: Antarc*; antarcticus
• Online Access: http://dx.doi.org/10.1128/aem.71.8.4225-4232.2005; https://journals.asm.org/doi/pdf/10.1128/AEM.71.8.4225-4232.2005

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-β- d -galactopyranoside) only below 30°C, an indication of cold-active β-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 β-galactosidase activity, belonged to glycoside hydrolase family 3, a family of β-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-β- d -glucopyranoside) than with the galactoside substrate ONPGal ( o -nitrophenyl-β- 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-β-glucosides such as helicin, MUG (4-methylumbelliferyl-β- d -glucopyranoside), esculin, indoxyl-β- 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 β-glucosidases. The hydrolysis of a variety of aryl-β-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low K m on indoxyl-β- d -glucoside may make it useful for producing indigo.