A new β-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization

Białkowska, Aneta; Cieśliński, Hubert; Nowakowska, Karolina Maria; Kur, Józef; Turkiewicz, Marianna

doi:10.1007/s00203-009-0509-4

Cited by 57 publications

(29 citation statements)

References 42 publications

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“…An optimal enzyme for this purpose would be active at pH 6.7-6.8 and at 4-8°C [51], which correlates with the pH profiles of the two identified β-galactosidases. Several cold-active β-galactosidases with the ability to hydrolyze lactose have been reported [45,49,50,52-54] and interestingly, recombinantly produced β-galactosidase from the Antarctic bacterium Pseudoalteromonas haloplanctis was able to outperform a commercial yeast enzyme at 4°C, where it retained around 20% of its activity even though the pH optimum was 8.5 [54]. The two enzymes from this study also show approximately 20% activity at 5°C and their pH optimum is around the pH of milk, suggesting that they could be good candidates for enzymatic hydrolysis of lactose.…”

Section: Resultsmentioning

confidence: 99%

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

2014

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BackgroundThe use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns.ResultsA strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities.ConclusionsMany cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low temperature, suggesting a possibly use in the dairy industry for this enzyme. The two different approaches complemented each other by targeting different microbial communities, highlighting the usefulness of combining methods for bioprospecting. Finally, we document here that ikaite columns constitute an important source of cold- and/or alkaline-active enzymes with industrial application potential.

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Section: Resultsmentioning

confidence: 99%

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

2014

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“…To date, studies indicate that cold-adapted enzymes typically display a high catalytic constant (k cat ), low optimal temperature, and high degree of thermolability, particularly of active center (14)(15)(16). A cold-adapted BG of the GH1 family, termed BglU, was previously cloned from the DNA library of Micrococcus antarcticus, a psychrophilic spherical bacterium found in Antarctica (17).…”

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confidence: 99%

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Miao

Hou

Fan

et al. 2016

Appl Environ Microbiol

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bPsychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic ␤-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (T m ). Mutation of His299 to Tyr increased the optimal temperature, the T m , and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.T he ␤-glucosidases (BGs; ␤-D-glucoside glycohydrolases, EC 3.2.1.21) are a widely occurring group of enzymes that cleave the carbohydrate moiety of short-chain oligosaccharides (two to six degrees of polymerization), alkyl, and aryl ␤-glucosides (1, 2). BGs have been classified into 135 glycoside hydrolase (GH) families, based on amino acid sequence similarity. GHs can also be classified into two major types, retaining and inverting enzymes, according to changes in anomeric configuration during hydrolytic reactions. Early studies of BGs were focused on their role in degrading the plant polymers cellulose and xylan (3, 4). More recently, these enzymes have gained commercial significance because of their biotechnological application in processes related to preparation of plant-based foods, e.g., conversion of phytoestrogen glucosides in fruits and vegetables to aglycone moieties, detoxification of cassava, and degradation of bitter compounds in citrus fruit juices and unripe olives (5, 6). Many mesophilic and thermophilic BGs have been cloned, purified, and characterized during the past 2 decades (5, 7-10). In contrast to mesophilic or thermophilic homologues, BGs from organisms adapted to cold...

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“…Several other cold-active ␤-galactosidases have been identified and characterized; these include those from Arthrobacter psychrolactophilus strain F2 (Nakagawa et al, 2007); Arthrobacter sp. 20B (Białkowska et al, 2009); and the psychrotolerant yeast Guehomyces pullulans 17-1 (Song et al, 2010). Some of these enzymes may be used in lactose hydrolysis in the future.…”

Section: Enzymes From Cold-adapted Organismsmentioning

confidence: 99%

The Role of Extremophilic Microorganisms and their Bioproducts in Food Processing and Production

Irwin

2012

Extremophiles

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A new β-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization

Cited by 57 publications

References 42 publications

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

The Role of Extremophilic Microorganisms and their Bioproducts in Food Processing and Production

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