Cloning, expression and structural stability of a cold-adapted β-galactosidase from Rahnella sp. R3

Fan, Yuting; Xiao, Hua; Zhang, Yuzhu; Yan, Feng; Shen, Qiuyun; Dong, Jun; Zhao, Wei; Zhang, Wenbin; Jin, Zhengyu; Yang, Ruijin

doi:10.1016/j.pep.2015.07.001

Cited by 37 publications

(30 citation statements)

References 24 publications

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“…Domain A shows highest similarity -by means of a Dali search (16) -to the catalytic domain from the GH42 βgalactosidase from Rahnella sp. (PDB: ID code 5E9A) with RMSD of 3.2 Å and complete query coverage (18). Overlay of these two structures revealed that the nucleophile (Glu314 in 5E9A) and acid/base residue (Glu157 in 5E9A) are conserved in Bs164 and the putative catalytic nucleophile and acid/base are Glu297 and Glu160 respectively.…”

Section: Crystal Structure Of Bs164 Reveals a Trimeric Quaternary Strmentioning

confidence: 99%

“…Domain B bears a striking resemblance to the trimerization domain of GH42 enzymes. In GH42 structures (15,18,20) there are a number of interactions between the trimerization domain and the catalytic domain which support trimeric quaternary structure. Likewise, domain B of Bs164 interacts over a surface area of 1380 Å 2 per protomer that includes seven residues involved in direct hydrogen bonds and two salt bridges between chains.…”

Section: Crystal Structure Of Bs164 Reveals a Trimeric Quaternary Strmentioning

confidence: 99%

“…Unlike the first two domains, the function of the C-terminal β-sandwich domain is much more difficult to infer from the structure. This domain is also present in GH42 enzymes (15,18,20) however, the role of this domain within GH42s is unknown.…”

Section: Crystal Structure Of Bs164 Reveals a Trimeric Quaternary Strmentioning

confidence: 99%

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Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

Armstrong

Davies

2020

Journal of Biological Chemistry

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Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-mannosidase activity, however, we conclusively show that it cleaves only β-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining β-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 Å resolution. Bs164 is composed of three domains: a (β/α)8 barrel, a trimerization domain, and a β-sandwich domain, representing a previously unobserved structural-fold for β-mannosidases. Structures of Bs164 at 1.80–2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.

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Section: Crystal Structure Of Bs164 Reveals a Trimeric Quaternary Strmentioning

confidence: 99%

Section: Crystal Structure Of Bs164 Reveals a Trimeric Quaternary Strmentioning

confidence: 99%

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Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

Armstrong

Davies

2020

Journal of Biological Chemistry

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“…A novel gene encoding a cold‐adapted β‐galactosidase (R‐β‐Gal) belonging to GH 42 from a psychrophilic gram‐negative bacterium Rahnella sp. R3 isolated and expressed in E. coli BL21 (DE3) by Fan et al (). In solution, the enzyme was found to be a homo‐trimer and was active at temperatures as low as 4°C while the purified enzyme displayed K m values of 6.5mM for ONPG and 2.2mM for lactose at 4°C.…”

Section: Cold‐active β‐Galactosidases From Psychrophilic and Psychrotmentioning

confidence: 99%

“…b-Galactosidase of Arthrobacter psychrophilus was active even at 08C, while some strains were capable of producing isozymes . isolated and expressed in E. coli BL21 (DE3) by Fan et al (2015). In solution, the enzyme was found to be a homo-trimer and was active at temperatures as low as 48C while the purified enzyme displayed K m values of 6.5 mM for ONPG and 2.2 mM for lactose at 48C.…”

Section: Recombinantmentioning

confidence: 99%

β-galactosidase: Biotechnological applications in food processing

2018

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β‐Galactosidases produced by microorganisms are being used in food technology for hydrolysis of lactose in milk and milk by‐products. The enzyme has attracted much attention in view of lactose intolerance in human population and due to importance of milk in human diet. β‐Galactosidase hydrolyze β‐galactopyranosides, that is, lactose, and form a range of trans‐galactosylation products or galactooligosaccharides (GOS) capable of providing several health benefits as prebiotics. In addition, the enzyme also finds applications in production of lactose based sweeteners from high lactose containing effluents of cheese manufacturing industries. Currently, the enzyme is mainly obtained from Aspergillus niger and Kluyveromyces lactis, however, they are thermolabile with optimum pH between 4.0 and 6.0 accompanied by low chemical resistance and high product inhibition properties. Thermostable enzymes are suitable to perform the hydrolytic process at relatively high temperatures, minimizing pathogen contamination, psychrophilic β‐galactosidases are preferred for treatment of milk under refrigerated conditions (<15°C), without heating and maintenance of temperature. Lactic acid bacteria (LAB) are a potential source of food grade enzymes and other metabolites. Although reports on the use of β‐galactosidase of LAB in pure form is reported for hydrolysis of lactose in milk and production of fermented milk products for reduced lactose content, methods of immobilization for use in continuous bioreactors are less explored. Recombinant DNA technology has attained much significance to reveal molecular aspects of enzyme catalysis, protein structure, gene organization for expression, and enhancement of thermophilic, psychrophilic, and mesophilic β‐galactosidases. Heterologous expression of extremozymes with β‐galactosidase would help in overcoming the limitations faced within the bioprocess technology. Practical applications Lactic acid bacteria (LAB) producing extracellular cell bound β‐galactosidase have been isolated and enzyme production was carried out using modified MRS medium incorporating lactose as enzyme inducer. Process for growing probiotic lactic acid bacteria for β‐galactosidase from fermented milk products has been patented (Indian Patent No. 247904). Thermostable β‐galactosidases were also extracted from Kluyveromyces lactis a yeast, isolated from curd samples and Bacillus sp. MTCC 864. An Indian patent has been granted for a simple, cost effective perforated two compartment immobilized enzyme bioreactor was designed and fabricated for lactose hydrolysis (Indian Patent No. 264609). The bioreactor can be operated continuously at temperature between 5.0 and 75.0°C, employing different immobilized enzyme preparations for different operational temperatures. β‐galactosidase of cell confined LAB and yeast have been immobilized in agarose and galactooligosaccharides (GOS) production was observed at 40% lactose concentration in 0.05M phosphate buffer at pH 6.5. Immobilized bioreactor preparation could be used for lactos...

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A novel β-galactosidase from Klebsiella oxytoca ZJUH1705 for efficient production of galacto-oligosaccharides from lactose

Huang

Zhu

Zhao

et al. 2020

Appl Microbiol Biotechnol

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Cloning, expression and structural stability of a cold-adapted β-galactosidase from Rahnella sp. R3

Cited by 37 publications

References 24 publications

Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

β-galactosidase: Biotechnological applications in food processing

A novel β-galactosidase from Klebsiella oxytoca ZJUH1705 for efficient production of galacto-oligosaccharides from lactose

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