Cold-Active β-Galactosidases: Insight into Cold Adaptation Mechanisms and Biotechnological Exploitation

Mangiagalli, Marco; Lotti, Marina

doi:10.3390/md19010043

Cited by 37 publications

(28 citation statements)

References 127 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Many biotechnology applications would benefit from the development of improved cold-active enzymes as they can be used to reduce the energy required to facilitate the same reaction, resulting in a greener process. 34,35 Development of a more cold-active β-galactosidase, specifically, may improve the production of lactose free milk, a process currently performed by a mesophilic enzyme at 20 C. 36 Cold-active enzymes may also be useful for many other catalytic processes which may be carried out at low temperatures. 37 The results presented here on a polyextremophilic enzyme confirm that a small number of residues are likely to be key to cold activity.…”

Section: Discussionmentioning

confidence: 99%

Double mutations far from the active site affect cold activity in an Antarctic halophilic β‐galactosidase

Laye

DasSarma

2022

Protein Science

View full text Add to dashboard Cite

The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for cold-active enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperaturedependent catalytic activity profiles via effects on K m and/or k cat compared to the wild-type enzyme. In this follow-up study, double-mutated enzymes were constructed and tested for temperature effects, including two new tandem residue pairs (N180T/A181T and T383A/S384A), and pairwise combination of the single residue mutations (N251D, F387L, I476V, and V482L). All doublemutated enzymes were found to be more catalytically active at moderate and/or less active at colder temperatures than wild-type, with both K m and k cat effects observed for the two tandem mutations. For pairwise combinations, a K m effect was seen when the surface exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface exposed mutation I476V located in a coiled region of domain B 25 Å from the active site was paired with N251D or V482L, a k cat effect was observed. These results indicate that temperature-dependent kinetic effects may be complex and subtle and are mediated by a combination of a small number of residues distant from the active site via changes to the hydration shell and/or perturbation of internal packing.

show abstract

Section: Discussionmentioning

confidence: 99%

Double mutations far from the active site affect cold activity in an Antarctic halophilic β‐galactosidase

Laye

DasSarma

2022

Protein Science

View full text Add to dashboard Cite

show abstract

“…As indicated by the gel filtration assay, native WspA1 should be a dimer and pH alteration cannot dissociate the dimer. Cold-active β-galactosidases are an attractive group identified in low temperature-adapted microorganisms 10 . Two cold-active β-galactosidases from Paracoccus sp.…”

Section: Discussionmentioning

confidence: 99%

“…The functionally active form of E. coli LacZ is a homotetramer with each monomer comprising of five structural domains, and the third (central) domain (residues 334–627) is an (α/β)8 barrel with an extended active-site cleft. Different sources of β-galactosidases differ in their optimum pH and temperature, thermal stability, substrate specificity, and metal ion cofactor sensitivity, providing a diversified selection for application in food processing 1 , 2 , 10 – 12 . Therefore, identification and characterization of new β-galactosidases from natural resources is beneficial for establishing glycosidase libraries and offers a wide variety of candidate glycosidases for application in food industry.…”

Section: Introductionmentioning

confidence: 99%

Characterization of two β-galactosidases LacZ and WspA1 from Nostoc flagelliforme with focus on the latter’s central active region

Gao

Liu

Cui

et al. 2021

Sci Rep

View full text Add to dashboard Cite

The identification and characterization of new β-galactosidases will provide diverse candidate enzymes for use in food processing industry. In this study, two β-galactosidases, Nf-LacZ and WspA1, from the terrestrial cyanobacterium Nostoc flagelliforme were heterologously expressed in Escherichia coli, followed by purification and biochemical characterization. Nf-LacZ was characterized to have an optimum activity at 40 °C and pH 6.5, different from that (45 °C and pH 8.0) of WspA1. Two enzymes had a similar Michaelis constant (Km = 0.5 mmol/liter) against the substrate o-nitrophenyl-β-D-galactopyranoside. Their activities could be inhibited by galactostatin bisulfite, with IC50 values of 0.59 µM for Nf-LacZ and 1.18 µM for WspA1, respectively. Gel filtration analysis suggested that the active form of WspA1 was a dimer, while Nf-LacZ was functional as a larger multimer. WspA1 was further characterized by the truncation test, and its minimum central region was found to be from residues 188 to 301, having both the glycosyl hydrolytic and transgalactosylation activities. Finally, transgenic analysis with the GFP reporter protein found that the N-terminus of WspA1 (35 aa) might play a special role in the export of WspA1 from cells. In summary, this study characterized two cyanobacterial β-galactosidases for potential applications in food industry.

show abstract

“…In addition to this, proteins and enzymes (referred to as cold-active enzymes or psychrozymes) are characterized by a more flexible 3D structure and high catalytic efficiency, necessary to avoid damages in the conformational state and gain better access to the substrate [112]. The functional properties of cold enzymes are due to several structural adaptations, including a peculiar aminoacidic composition (mainly a higher Glu+Asp/Arg+Lys ratio than mesophilic enzymes), reduced amount of some salt bridge-forming amino acids [113,114], presence of weak intramolecular bound interactions (including hydrogen bonds, electrostatic and salt bridges), higher surface hydrophobicity level and negative charge, and reduction in metal binding affinity [115]. All parameters distinguishing psychroenzymes from mesophilic or thermophilic enzymes have been widely reviewed by Mangiagalli and Lotti [115], who substantially argued that their true hallmark is the ability to maintain high activity at low temperature, and to adapt their oligomerization, flexibility, and plasticity to ensure catalyzation of reactions at low temperature and substrate promiscuity.…”

Section: Microbial Diversity In Cryoenvironmentsmentioning

confidence: 99%

Life from a Snowflake: Diversity and Adaptation of Cold-Loving Bacteria among Ice Crystals

Rizzo

Giudice

2022

Crystals

View full text Add to dashboard Cite

Incredible as it is, researchers have now the awareness that even the most extreme environment includes special habitats that host several forms of life. Cold environments cover different compartments of the cryosphere, as sea and freshwater ice, glaciers, snow, and permafrost. Although these are very particular environmental compartments in which various stressors coexist (i.e., freeze–thaw cycles, scarce water availability, irradiance conditions, and poorness of nutrients), diverse specialized microbial communities are harbored. This raises many intriguing questions, many of which are still unresolved. For instance, a challenging focus is to understand if microorganisms survive trapped frozen among ice crystals for long periods of time or if they indeed remain metabolically active. Likewise, a look at their site-specific diversity and at their putative geochemical activity is demanded, as well as at the equally interesting microbial activity at subzero temperatures. The production of special molecules such as strategy of adaptations, cryoprotectants, and ice crystal-controlling molecules is even more intriguing. This paper aims at reviewing all these aspects with the intent of providing a thorough overview of the main contributors in investigating the microbial life in the cryosphere, touching on the themes of diversity, adaptation, and metabolic potential.

show abstract

Cold-Active β-Galactosidases: Insight into Cold Adaptation Mechanisms and Biotechnological Exploitation

Cited by 37 publications

References 127 publications

Double mutations far from the active site affect cold activity in an Antarctic halophilic β‐galactosidase

Double mutations far from the active site affect cold activity in an Antarctic halophilic β‐galactosidase

Characterization of two β-galactosidases LacZ and WspA1 from Nostoc flagelliforme with focus on the latter’s central active region

Life from a Snowflake: Diversity and Adaptation of Cold-Loving Bacteria among Ice Crystals

Contact Info

Product

Resources

About