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A Review on Psychrophilic β-D-Galactosidases and Their Potential Applications

Reddy LJ, Kumar PS, Pandrangi SL, Chikati R, Srinivasulu C, John A, et al.

Appl Biochem Biotechnol, 2023 Apr;195(4):2743-2766.
PMID: 36422804 DOI: 10.1007/s12010-022-04215-w

The majority of the Earth's ecosystem is frigid and frozen, which permits a vast range of microbial life forms to thrive by triggering physiological responses that allow them to survive in cold and frozen settings. The apparent biotechnology value of these cold-adapted enzymes has been targeted. Enzymes' market size was around USD 6.3 billion in 2017 and will witness growth at around 6.8% CAGR up to 2024 owing to shifting consumer preferences towards packaged and processed foods due to the rising awareness pertaining to food safety and security reported by Global Market Insights (Report ID-GMI 743). Various firms are looking for innovative psychrophilic enzymes in order to construct more effective biochemical pathways with shorter reaction times, use less energy, and are ecologically acceptable. D-Galactosidase catalyzes the hydrolysis of the glycosidic oxygen link between the terminal non-reducing D-galactoside unit and the glycoside molecule. At refrigerated temperature, the stable structure of psychrophile enzymes adjusts for the reduced kinetic energy. It may be beneficial in a wide variety of activities such as pasteurization of food, conversion of biomass, biological role of biomolecules, ambient biosensors, and phytoremediation. Recently, psychrophile enzymes are also used in claning the contact lens. β-D-Galactosidases have been identified and extracted from yeasts, fungi, bacteria, and plants. Conventional (hydrolyzing activity) and nonconventional (non-hydrolytic activity) applications are available for these enzymes due to its transgalactosylation activity which produce high value-added oligosaccharides. This review content will offer new perspectives on cold-active β-galactosidases, their source, structure, stability, and application.

Matched MeSH terms: beta-Galactosidase/metabolism
Papaya beta-galactosidase/galactanase isoforms in differential cell wall hydrolysis and fruit softening during ripening

Lazan H, Ng SY, Goh LY, Ali ZM

Plant Physiol Biochem, 2004 Dec;42(11):847-53.
PMID: 15694277

The potential significance of the previously reported papaya (Carica papaya L.) beta-galactosidase/galactanase (beta-d-galactoside galactohydrolase; EC 3.2.1.23) isoforms, beta-gal I, II and III, as softening enzymes during ripening was evaluated for hydrolysis of pectins while still structurally attached to unripe fruit cell wall, and hemicelluloses that were already solubilized in 4 M alkali. The enzymes were capable of differentially hydrolyzing the cell wall as evidenced by increased pectin solubility, pectin depolymerization, and degradation of the alkali-soluble hemicelluloses (ASH). This enzyme catalyzed in vitro changes to the cell walls reflecting in part the changes that occur in situ during ripening. beta-Galactosidase II was most effective in hydrolyzing pectin, followed by beta-gal III and I. The reverse appeared to be true with respect to the hemicelluloses. Hemicellulose, which was already released from any architectural constraints, seemed to be hydrolyzed more extensively than the pectins. The ability of the beta-galactanases to markedly hydrolyze pectin and hemicellulose suggests that galactans provide a structural cross-linkage between the cell wall components. Collectively, the results support the case for a functional relevance of the papaya enzymes in softening related changes during ripening.

Matched MeSH terms: beta-Galactosidase/metabolism*
Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides

Balasubramaniam S, Lee HC, Lazan H, Othman R, Ali ZM

Phytochemistry, 2005 Jan;66(2):153-63.
PMID: 15652572

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.

Matched MeSH terms: beta-Galactosidase/metabolism*
Dendrimer-like nanoparticles based β-galactosidase assembly for enhancing its selectivity toward transgalactosylation

Misson M, Du X, Jin B, Zhang H

Enzyme Microb Technol, 2016 Mar;84:68-77.
PMID: 26827776 DOI: 10.1016/j.enzmictec.2015.12.008

Functional nanomaterials have been pursued to assemble nanobiocatalysts since they can provide unique hierarchical nanostructures and localized nanoenvironments for enhancing enzyme specificity, stability and selectivity. Functionalized dendrimer-like hierarchically porous silica nanoparticles (HPSNs) was fabricated for assembling β-galactosidase nanobiocatalysts for bioconversion of lactose to galacto-oligosaccharides (GOS). The nanocarrier was functionalized with amino (NH2) and carboxyl (COOH) groups to facilitate enzyme binding, benchmarking with non-functionalized HPSNs. Successful conjugation of the functional groups was confirmed by FTIR, TGA and zeta potential analysis. HPSNs-NH2 showed 1.8-fold and 1.1-fold higher β-galactosidase adsorption than HPSNs-COOH and HPSNs carriers, respectively, with the highest enzyme adsorption capacity of 328mg/g nanocarrier at an initial enzyme concentration of 8mg/ml. The HPSNs-NH2 and β-galactosidase assembly (HPSNs-NH2-Gal) demonstrated to maintain the highest activity at all tested enzyme concentrations and exhibited activity up to 10 continuous cycles. Importantly, HPSNs-NH2-Gal was simply recycled through centrifugation, overcoming the challenging problems of separating the nanocarrier from the reaction medium. HPSNs-NH2-Gal had distinguished catalytic reaction profiles by favoring transgalactosylation, enhancing GOS production of up to 122g/l in comparison with 56g/l by free β-galactosidase. Furthermore, it generated up to 46g/l GOS at a lower initial lactose concentration while the free counterpart had negligible GOS production as hydrolysis was overwhelmingly dominant in the reaction system. Our research findings show the amino-functionalized HPSNs can selectively promote the enzyme activity of β-galactosidase for transgalactosylation, which is beneficial for GOS production.

Matched MeSH terms: beta-Galactosidase/metabolism*
Fulltext Differential expression of galectin-3 in advancing thyroid cancer cells: a clue toward understanding tumour progression and metastasis

Htwe TT, Karim N, Wong J, Jahanfar S, Mansur MA

Singapore Med J, 2010 Nov;51(11):856-9.
PMID: 21140111

INTRODUCTION: Galectin-3 is a member of the beta-galactoside-binding protein family that plays an important role in cell-to-cell adhesion and in cell-to-matrix interaction. Cellular expression of galectin-3 is correlated with cancer aggressiveness and metastasis.
METHODS: We examined the differential expression of galectin-3 in a collection of 142 cases of thyroid lesions, including 108 cases of papillary thyroid carcinoma (PTC) and 34 cases of follicular carcinoma (FCA). An immunohistochemical method was applied and semiquantitative scoring was performed on the staining intensity of the positive tissue. Scoring was done on cells at the central portion of the tumour foci and on cells at the periphery that were adjacent to the neighbouring normal thyroid tissue matrix.
RESULTS: A significantly higher expression (p is 0.001) of galectin-3 was observed in the advancing peripheral thyroid cancer cells compared to the centrally located cells that were not in close contact with the neighbouring stromal tissue in cases with PTC compared to those with FCA.
CONCLUSION: This finding supported the role of galectin-3 in its cell-to-cell adhesion and cell-to-matrix interaction. Galectin-3 is a potential tumour marker for indicating local and distance metastasis, especially in cases with PTC.

Matched MeSH terms: beta-Galactosidase/metabolism
Fulltext Reversal of myoblast aging by tocotrienol rich fraction posttreatment

Lim JJ, Ngah WZ, Mouly V, Abdul Karim N

Oxid Med Cell Longev, 2013;2013:978101.
PMID: 24349615 DOI: 10.1155/2013/978101

Skeletal muscle satellite cells are heavily involved in the regeneration of skeletal muscle in response to the aging-related deterioration of the skeletal muscle mass, strength, and regenerative capacity, termed as sarcopenia. This study focused on the effect of tocotrienol rich fraction (TRF) on regenerative capacity of myoblasts in stress-induced premature senescence (SIPS). The myoblasts was grouped as young control, SIPS-induced, TRF control, TRF pretreatment, and TRF posttreatment. Optimum dose of TRF, morphological observation, activity of senescence-associated β-galactosidase (SA-β-galactosidase), and cell proliferation were determined. 50 μg/mL TRF treatment exhibited the highest cell proliferation capacity. SIPS-induced myoblasts exhibit large flattened cells and prominent intermediate filaments (senescent-like morphology). The activity of SA-β-galactosidase was significantly increased, but the proliferation capacity was significantly reduced as compared to young control. The activity of SA-β-galactosidase was significantly reduced and cell proliferation was significantly increased in the posttreatment group whereas there was no significant difference in SA-β-galactosidase activity and proliferation capacity of pretreatment group as compared to SIPS-induced myoblasts. Based on the data, we hypothesized that TRF may reverse the myoblasts aging through replenishing the regenerative capacity of the cells. However, further investigation on the mechanism of TRF in reversing the myoblast aging is needed.

Matched MeSH terms: beta-Galactosidase/metabolism
Fulltext Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

Makpol S, Yeoh TW, Ruslam FA, Arifin KT, Yusof YA

BMC Complement Altern Med, 2013;13:210.
PMID: 23948056 DOI: 10.1186/1472-6882-13-210

Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase.

Matched MeSH terms: beta-Galactosidase/metabolism
The impact of long-term in vitro expansion on the senescence-associated markers of human adipose-derived stem cells

Safwani WK, Makpol S, Sathapan S, Chua KH

Appl Biochem Biotechnol, 2012 Apr;166(8):2101-13.
PMID: 22391697 DOI: 10.1007/s12010-012-9637-4

Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.

Matched MeSH terms: beta-Galactosidase/metabolism
Fulltext Tocotrienol-rich fraction prevents cell cycle arrest and elongates telomere length in senescent human diploid fibroblasts

Makpol S, Durani LW, Chua KH, Mohd Yusof YA, Ngah WZ

J Biomed Biotechnol, 2011;2011:506171.
PMID: 21541185 DOI: 10.1155/2011/506171

This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs). Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G(0)/G(1) phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G(0)/G(1) phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.

Matched MeSH terms: beta-Galactosidase/metabolism
Evaluation of morphological changes of Staphylococcus aureus and Escherichia coli induced with the antimicrobial peptide AN5-1

Alkotaini B, Anuar N, Kadhum AA

Appl Biochem Biotechnol, 2015 Feb;175(4):1868-78.
PMID: 25427593 DOI: 10.1007/s12010-014-1410-4

The mechanisms of action of AN5-1 against Gram-negative and Gram-positive bacteria were investigated by evaluations of the intracellular content leakage and by microscopic observations of the treated cells. Escherichia coli and Staphylococcus aureus were used for this investigation. Measurements of DNA, RNA, proteins, and β-galactosidase were taken, and the results showed a significant increase in the cultivation media after treatment with AN5-1 compared with the untreated cells. The morphological changes of treated cells were shown using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The observations showed that AN5-1 acts against E. coli and against S. aureus in similar ways, by targeting the cell wall, causing disruptions; at a high concentration (80 AU/ml), these disruptions led to cell lysis. The 3D AFM imaging system showed that at a low concentration of 20 AU/ml, the effect of AN5-1 is restricted to pore formation only. Moreover, a separation between the cell wall and the cytoplasm was observed when Gram-negative bacteria were treated with a low concentration (20 AU/ml) of AN5-1.

Matched MeSH terms: beta-Galactosidase/metabolism
Fulltext Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

Zainuddin A, Chua KH, Abdul Rahim N, Makpol S

BMC Mol. Biol., 2010;11:59.
PMID: 20707929 DOI: 10.1186/1471-2199-11-59

Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H2O2-induced oxidative stress and gamma-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.

Matched MeSH terms: beta-Galactosidase/metabolism
Fulltext Postharvest analysis of lowland transgenic tomato fruits harboring hpRNAi-ACO1 construct

Behboodian B, Mohd Ali Z, Ismail I, Zainal Z

ScientificWorldJournal, 2012;2012:439870.
PMID: 22919320 DOI: 10.1100/2012/439870

The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding to ACO1 of lowland tomato cultivar (MT1), which has high identity with ACO1 of Solanum lycopersicum in GenBank, was cloned through RT-PCR. Using a partial coding region of ACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1 construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms of β-galactosidase (β-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid.

Matched MeSH terms: beta-Galactosidase/metabolism
Fulltext The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts

Khor SC, Razak AM, Wan Ngah WZ, Mohd Yusof YA, Abdul Karim N, Makpol S

PLoS One, 2016;11(2):e0149265.
PMID: 26885980 DOI: 10.1371/journal.pone.0149265

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.

Matched MeSH terms: beta-Galactosidase/metabolism
Isolation, Expansion, and Characterization of Wharton's Jelly-Derived Mesenchymal Stromal Cell: Method to Identify Functional Passages for Experiments

Aung SW, Abu Kasim NH, Ramasamy TS

Methods Mol Biol, 2019;2045:323-335.
PMID: 31201682 DOI: 10.1007/7651_2019_242

The therapeutic potential of human mesenchymal stromal stem cells (hMSCs) for cell-based therapeutic is greatly influenced by the in vitro culture condition including the culture conditions. Nevertheless, there are many technical challenges needed to be overcome prior to the clinical use including the quantity, quality, and heterogeneity of the cells. Therefore, it is necessary to develop a stem cell culture procedure or protocol for cell expansion in order to generate reproducible and high-quality cells in accordance with good manufacturing practice for clinical and therapeutic purposes. Here we assessed the MSCs characteristic of human Wharton's jelly mesenchymal stromal cells in in vitro culture according to the criteria established by the International Society for Cellular Therapy. Besides, the viability of the WJMSCs was determined in order to increase the confidence that the cells are employed to meet the therapeutic efficacy.

Matched MeSH terms: beta-Galactosidase/metabolism
Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts

Makpol S, Zainuddin A, Chua KH, Yusof YA, Ngah WZ

Clinics (Sao Paulo), 2012;67(2):135-43.
PMID: 22358238

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes.
METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer.
RESULTS: The cell cycle was arrested in the G(0)/G(1) phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G(0)/G(1) phase and increased cell populations in the G(2)/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts.
CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

Matched MeSH terms: beta-Galactosidase/metabolism

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