Thermostable Β-galactosidase Research Articles

Isolation and characterization of a β-galactosidase from Lactobacillus helveticus for industrial processing

In this study, a thermostable β-galactosidase from Lactobacillus helveticus (Lb. helveticus) OSU-PECh-4A has been isolated through diafiltration and size-exclusion chromatography. The enzyme consists of a heterodimer with a molecular mass of 110 kDa, with a small and large subunit of 36 kDa and 74 kDa, respectively. The Km and Vmax values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) hydrolysis were, respectively, 29.87 ± 1.05 mM, 1.88 ± 0.02 μmol D-glucose released per min per mg of protein, and 0.067 ± 0.003 mM, 1.70 ± 0.05 μmol o-nitrophenol (oNP) released per min per mg of protein. This β-galactosidase is significantly activated by Mg+2 (2–10 mM) and slightly inhibited by D-Glucose. The enzyme can also hydrolyze 57 ± 3% of lactose after 12 h of reaction at 45°C and under high lactose concentration. We propose that this enzyme provides a significant advantage from a practical and consumer point of view due to its origins as a probiotic source and improved features for important industrial applications, such as lactose hydrolysis and the potential to produce galacto-oligosaccharides (GOS).

Production of Daidzein and Genistein from Seed and Root Extracts of Korean Wild Soybean (Glycine soja) by Thermostable β-Galactosidase from Thermoproteus uzoniensis

Isoflavone glycosides are commonly biotransformed into isoflavone aglycones due to the superior biological activities of the latter. Wild soybeans contain a higher isoflavone content than domesticated soybeans due to their high level of genetic diversity. In this study, we cloned and characterized a thermostable β-galactosidase from the extreme thermophile Thermoproteus uzoniensis for potential application in isoflavone conversion in Korean wild soybeans. The purified recombinant enzyme exhibited a maximum specific activity of 1103 μmol/min/mg at pH 5.0 and 90 °C with a half-life of 46 h and exists as a homodimer of 113 kDa. The enzyme exhibited the highest activity for p-nitrophenyl (pNP)-β-D-galactopyranoside among aryl glycosides and it hydrolyzed isoflavone glycosides in the order genistin > daidzin > ononin > glycitin. The enzyme completely hydrolyzed 2.77 mM daidzin and 3.85 mM genistin in the seed extract of wild soybean after 80 and 70 min with productivities of 1.86 and 3.30 mM/h, respectively, and 9.89 mM daidzin and 1.67 mM genistin in the root extract after 180 and 30 min, with the highest productivities of 3.30 and 3.36 mM/h, respectively, compared to other glycosidases. Our results will contribute to the industrial production of isoflavone aglycone using wild soybean and this is the first report on the enzymatic production of isoflavone aglycones from isoflavone glycosides in wild soybeans.

A Novel Thermal-Activated β-Galactosidase from Bacillus aryabhattai GEL-09 for Lactose Hydrolysis in Milk.

β-Galactosidase has been greatly used in the dairy industry. This study investigated a novel thermostable β-galactosidase (lacZBa) from Bacillus aryabhattai GEL-09 and evaluated the hydrolytic performance of this enzyme. Firstly, the lacZBa-encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). Phylogenetic analyses revealed that lacZBa belonged to the glycoside hydrolase family 42. Using SDS-PAGE, we determined that the molecular weight of lacZBa was ~75 kDa. Purified lacZBa exhibited a maximum activity at 45 °C, pH 6.0, and could be activated following incubation at 45 °C for several minutes. The half-life of lacZBa at 45 °C and 50 °C was 264 h and 36 h, respectively. While Co2+, Mn2+, Zn2+, Fe2+, Mg2+, and Ca2+ enhanced enzymatic activity, Cu2+ and ethylenediaminetetraacetic acid inhibited enzymatic activity. Moreover, lacZBa could hydrolyze lactose and oNPG with Km values of 85.09 and 14.38 mM. Molecular docking results revealed that lacZBa efficiently recognized and catalyzed lactose. Additionally, the hydrolysis of lactose by lacZBa was studied in lactose solution and commercial milk. Lactose was completely hydrolyzed within 4 h with 8 U/mL of lacZBa at 45 °C. These results suggested that lacZBa identified in this study has potential applications in the dairy industry.

Characterization of a Thermally Stable β-galactosidase Produced by Thermophilic Anoxybacillus sp. AH1

Thermostable β-galactosidases from thermophilic bacteria have attracted increasing interest to have various advantages in industrial and biotechnological applications. In this study, a highly thermally stable β-galactosidase produced by Anoxybacillus sp. AH1was purified and characterized. The highest enzyme production was achieved after the bacterium was incubated for 24 hours. The enzyme was purified by precipitation with ammonium sulphate dialysis, gel filtration chromatography using Sephadex G-75. After the purification steps, β-galactosidase was found to be purified 10.2-fold and a yield of 13.9%. The molecular mass of the galactosidase was estimated to be 75 kDa by SDS-PAGE. The purified enzyme was highly stable and retained at 71% of the original activity at 60 °C and 53% at 70 oC within 120 minutes. The Km and Vmax values of purified β-galactosidase were calculated as 1.249 mM and 0.5 μmol minutes-1, respectively. Ca2+, Zn2+, and Mg2+ significantly activated β-galactosidase activity, whereas enzyme activity was inhibited significantly by Cu+2 as well as by the metal ion chelators1,10-phenanthroline (phen) and ethylenediaminetetraacetic acid (EDTA). The Purified β-galactosidase activity was increased by PMSF (phenylmethylsulfonyl fluoride), PCMB (p-chloromercuribenzoic acid), DTT (dithiothreitol), and β-ME (β-mercaptoethanol) at 2 mM, but inhibited completely by NEM (N-ethylmaleimide) at 1 mM.

Freeze-thaw system for thermostable β-Galactosidase isolation from Gedong Songo Geobacillus sp. isolate

The effective isolation of intracellular enzymes from thermophilic bacteria is challenging because of their sturdy membrane. On the other hand, the low-cost and nontoxic method is essential for industrial food enzymes. The freeze-thaw cycles using acetone-dry ice as a frozen system was studied for efficient isolation of thermostable b-galactosidase from Geobacillus sp. dYTae-14. This enzyme has been known for application in the dairy industry to reduce the lactose content. In this study, the freeze-thaw method was performed with cycle variations 3, 5, and 7 cycles. Acetone-dry ice (-78°C) is used as a frozen system and boiling water for thawing. The b-galactosidase activity was assayed using ortho-Nitrophenyl-β-galactoside (ONPG) as substrate and protein content determined with the Lowry method. The results show that the most effective freeze-thaw is five cycles. The enzyme’s highest specific activity is 3610.13 units/mg proteins at 40-60 % ammonium sulfate saturation, with a purity value of 2.52.

Effect of biopolymer matrices on lactose hydrolysis by enzymatically active hydrogel and aerogels loaded with β-galactosidase nanoflowers

In this work, enzymatically active polysaccharide-based hydrogels and aerogels have been developed. To this end, a thermostable β-galactosidase (TmLac) enzyme from Thermotoga maritima embedded in nanoflowers’ format was used to evaluate the capacity of the hydrogel matrices to preserve the hydrolytic activity of the enzyme and the reusability of the hydrogels formed. Commercial agar, unpurified agar and agarose were compared as supporting materials. Although the developed hydrogel capsules can be used at high temperature (75 °C) and reused for the digestion of lactose to a greater extent than the free nanoflowers, loaded hydrogel capsules behaved differently depending on the type of polysaccharide used. Commercial agar was the most promising one since these hydrogel capsules could be reused, maintaining the structural integrity and reaching higher enzymatic activity (after seven cycles at 75 °C) than the free TmLac-Ca2+ nanoflowers.To facilitate handling and storage, aerogels were developed by freeze-drying the hydrogel capsules. Aerogels of agarose and unpurified agar underwent structural changes during freeze-drying that adversely affected their subsequent use, losing their integrity after being rehydrated. However, commercial agar aerogels were successfully developed and reused thanks to the existing interactions with TmLac-Ca2+ nanoflowers (confirmed by FTIR), which resulted in better capsule integrity and enzyme protection. The hydrolytic activity of enzymatically active aerogels based on commercial agar was in the same range of the free TmLac and TmLac-Ca2+ nanoflowers, being significantly higher to their counterparts in the hydrated form (hydrogels based on commercial agar).

Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

BackgroundThermostable enzymes are commonly produced in mesophilic hosts for research and bioengineering purposes. However, these hosts do not overexpress the active forms of some biologically functional thermoenzymes. Therefore, an efficient thermophilic expression system is needed. Thermus thermophilus contains an easily manipulable genome and is therefore among the best candidate microbes for a “hot” expression system. We previously identified a strong and inducible promoter that was active in T. thermophilus under supersaturated silica conditions. Here, we report a new heterologous gene expression system based on a silica-inducible promoter in T. thermophilus.ResultsA Thermus sp. A4 gene encoding thermostable β-galactosidase was cloned as a reporter gene into the expression vector pSix1, which contains a selection marker that confers thermostable resistance to hygromycin and a 600 bp DNA region containing a putative silica-inducible promoter. β-galactosidase activity was 11-fold higher in the presence than in the absence of 10 mM silicic acid. SDS-PAGE revealed a prominent band corresponding to 73 kDa of β-galactosidase, and this enzyme was expressed as an active and soluble protein (yield: 27 mg/L) in Thermus but as an inclusion body in Escherichia coli. Truncation of the putative silica-inducible promoter region in Thermus expression vector improved the yield of the target protein, possibly by avoiding plasmid instability due to homologous recombination. Finally, we developed an expression vector containing the pSix1 backbone and a 100 bp DNA region corresponding to the silica-inducible promoter. We used this vector to successfully express the active form of glutamate dehydrogenase from Pyrobaculum islandicum (PisGDH) without additional treatment (yield: 9.5 mg/L), whereas the expression of active PisGDH in E. coli required heat treatment.ConclusionWe successfully expressed the thermostable β-galactosidase and PisGDH in T. thermophilus as active and soluble forms and achieved with our system the highest known protein expression levels in this species. These thermoenzymes were expressed in active and soluble forms. Our results validate the use of our silica-inducible expression system as a novel strategy for the intracellular overexpression of thermostable proteins.

Codon optimization and co-expression of thermostable β-galactosidase and L-arabinose isomerase in lactococcus lactis for single-step production of food-grade D-tagatose

Codon optimization and co-expression of thermostable β-galactosidase and L-arabinose isomerase in lactococcus lactis for single-step production of food-grade D-tagatose

Recyclable thermophilic hybrid protein-inorganic nanoflowers for the hydrolysis of milk lactose

Thermostable β-galactosidase (TmLac) has been immobilized as hybrid inorganic-protein nanoflowers using salts of Cu2+, Mn2+, Zn2+, Co2+ and Ca2+ as the inorganic component. The incorporation efficiency of enzyme into the nanoflowers was higher than 95% for a protein concentration of 0.05 mg/mL. The structure, activity and recyclability of the nanoflowers with different chemical composition were analyzed. Ca2+, Mn2+ and Co2+ nanoflowers showed a level of lactase activity equivalent to their same content of free enzyme. Cu2+nanoflowers showed only marginal enzyme activity in agreement with the inhibitory effect of this cation on the enzyme. TmLac nanoflowers provide an efficient methodology for enzyme immobilization and recyclability. TmLac-Ca2+ nanoflowers presented the best properties for lactose hydrolysis both in buffered and in milk, and could be reused in five consecutive cycles.

Simulation and economic assessment of large-scale enzymatic N-acetyllactosamine manufacture

N-acetyllactosamine (LacNAc) is an important lactose-derived molecule which can act as an effective prebiotic. In this study a process for the enzymatic synthesis and downstream purification of LacNAc was designed based on the use of thermostable β-galactosidases from Bacillus circulans (BgaD-D), Thermus thermophilus HB27 or Pyrococcus furiosus (CelB) respectively. Four configurations for the purification stage were simulated; anion-exchange chromatography, an activated charcoal-Celite column, N-acetylglucosamine (GlcNAc) crystallization and an activated charcoal-Celite column, as well as selective crystallization. While the enzyme CelB has greater stability at higher temperatures, this enzyme gives a lower LacNAc yield, leading to significant capital investment. For the design based on the BgaD-D biocatalyst and anion exchange chromatography, recovery of GlcNAc improved the project profitability when the GlcNAc price was greater than $10 per kg. GlcNAc was the main contributor to the raw material costs for most processes, although methanol contributed 72% of these costs for the process based on an activated charcoal column. The use of a crystallizer for GlcNAc separation before this column, reduced this methanol consumption by 73%. The use of selective crystallization proved the best approach, reducing the minimum LacNAc sales price to $2 per gram. The plant was more economic when the acceptor to donor ratio was reduced from 10 to 4 and the lactose concentration increased from 50 mM to 550 mM.

A novel thermostable β-galactosidase from Bacillus coagulans with excellent hydrolysis ability for lactose in whey

β-Galactosidase is one of the most important enzymes used in dairy industry. Here, a novel thermostable β-galactosidase was cloned and overexpressed from Bacillus coagulans NL01 in Escherichia coli. The phylogenetic trees were constructed using neighbor-joining methods. Phylogeny and amino acid analysis indicated that this enzyme belonged to family 42 of glycoside hydrolases. The optimal pH and temperature were, respectively, 6.0 and 55 to 60°C. The purified enzyme had a 3.5-h half-life at 60°C. Enzyme activity was enhanced by Mn2+. Compared with other β-galactosidases from glycoside hydrolase family 42, B. coagulans β-galactosidase exhibited excellent hydrolysis activity. The Michaelis constant (Km) and maximum rate of enzymatic reaction (Vmax) values for p-nitrophenyl-β-d-galactopyranoside and o-nitrophenyl-β-d-galactopyranoside were 1.06 mM, 19,383.60 U/mg, and 2.73 mM, 5,978.00 U/mg, respectively. More importantly, the enzyme showed lactose hydrolysis ability superior to that of the commercial enzyme. The specific enzyme activity for lactose was 27.18 U/mg. A total of 104.02 g/L lactose in whey was completely hydrolyzed in 3 h with addition of 2.38 mg of pure enzyme per gram of lactose. In view of the high price of commercial β-galactosidase, B. coagulans β-galactosidase could be a promising prototype for development of commercial enzymes aimed at lactose treatment in the dairy industry.

Matryoshka enzyme encapsulation: Development of zymoactive hydrogel particles with efficient lactose hydrolysis capability.

This report describes an efficient procedure for enzyme encapsulation and its application for the hydrolysis of lactose. The enzymatic material that has been developed consists of hydrogel particles (ca. 3–4 mm of diameter) composed of either alginate or an alginate-agarose combination, in which bacterial cells loaded with a thermostable β-galactosidase are embedded. The cells were rendered fully permeable to the substrate, either chromogenic p-nitrophenyl galactose or lactose, by thermal treatment at 75 °C. Hydrogel particles made of a mixture of alginate and agarose displayed high catalytic activity (i.e. 1 g of beads hydrolyze the lactose equivalent of 100 mL of milk in 15 min) and thermal stability: they could be reused at 75 °C for complete hydrolysis of 5% (w/v) lactose solution at least six consecutive times without significant loss of activity.

Heat-Loving β-Galactosidases from Cultured and Uncultured Microorganisms.

β-galactosidases (EC.3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in the food industry: the production of low-lactose milk and dairy goods for lactose intolerant people, and the generation of galacto-oligosaccharides by transgalactosylation reactions. Due to their thermostability, β-galactosidases from thermophilic microorganisms are very interesting for industrial processes, as high temperatures can increase the initial productivity of the enzyme, provide higher solubility of substrates, and prevent microbial contamination. In the past, it was necessary to cultivate and grow thermophilic microorganisms to discover novel thermozymes, but the development of metagenomic techniques has allowed researchers to access the genomic potential of uncultivated microbes and their enzymes. The present review gives a brief outline of thermophilic β-galactosidases, with a special focus on those obtained through metagenomics. Additionally, the sequences of β-galactosidases found in some public metagenomes from hot springs were studied and compared to other known thermostable β-galactosidases.

Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications.

The use of Bacillus subtilis in synthetic biology and metabolic engineering is highly desirable to take advantage of the unique metabolic pathways present in this organism. To do this, an evaluation of B. subtilis’ intrinsic biological parts is required to determine the best strategies to accurately regulate metabolic circuits and expression of target proteins. The strengths of promoter candidates were evaluated by measuring relative fluorescence units of a green fluorescent protein reporter, integrated into B. subtilis’ chromosome. A total of 84 predicted promoter sequences located upstream of different classes of proteins including heat shock proteins, cell-envelope proteins, and proteins resistant against toxic metals (based on similarity) and other kinds of genes were tested. The expression levels measured ranged from 0.0023 to 4.53-fold of the activity of the well-characterized strong promoter P43. No significant shifts were observed when strains, carrying different promoter candidates, were cultured at high temperature or in media with ethanol, but some strains showed increased activity when cultured under high osmotic pressure. Randomly selected promoter candidates were tested and found to activate transcription of thermostable β-galactosidase (bgaB) at a similar level, implying the ability of these sequences to function as promoter elements in multiple genetic contexts. In addition, selected promoters elevated the final production of both cytoplasmic bgaB and secreted protein α-amylase to about fourfold and twofold, respectively. The generated data allows a deeper understanding of B. subtilis’ metabolism and will facilitate future work to develop this organism for synthetic biology.

Thermostable β-galactosidases for the synthesis of human milk oligosaccharides

Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk. Using lactose as a cheap donor, some β-galactosidases (EC 3.2.1.23) can catalyze transgalactosylation to form the human milk oligosaccharide lacto-N-neotetraose (LNnT; Gal-β(1,4)-GlcNAc-β(1,3)-Gal-β(1,4)-Glc). In order to reduce reaction times and be able to work at temperatures, which are less welcoming to microbial growth, the current study investigates the possibility of using thermostable β-galactosidases for synthesis of LNnT and N-acetyllactosamine (LacNAc; Gal-β(1,4)-GlcNAc), the latter being a core structure in HMOs. Two hyperthermostable GH 1 β-galactosidases, Ttβ-gly from Thermus thermophilus HB27 and CelB from Pyrococcus furiosus, were codon-optimized for expression in Escherichia coli along with BgaD-D, a truncated version of the GH 42 β-galactosidase from Bacillus circulans showing high transgalactosylation activity at low substrate concentrations. The three β-galactosidases were compared in the current study in terms of their transgalactosylation activity in the formation of LacNAc and LNnT. In all cases, BgaD-D was the most potent transgalactosidase, but both thermostable GH 1 β-galactosidases could catalyze formation of LNnT and LacNAc, with Ttβ-gly giving higher yields than CelB. The thermal stability of the three β-galactosidases was elucidated and the results were used to optimize the reaction efficiency in the formation of LacNAc, resulting in 5-6 times higher reaction yields and significantly shorter reaction times.

A new potential secretion pathway for recombinant proteins in Bacillus subtilis.

BackgroundSecretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable ‘cell factory’ for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium.ResultsAll four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides.ConclusionOur results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0374-6) contains supplementary material, which is available to authorized users.

Gene clone and characterization of a novel thermostable β-galactosidase with transglycosylation activity from Thermotoga naphthophila RUK-10

We cloned and expressed a new recombinant β-galactosidase(TN0949) from Thermotoga naphthophila RKU-10 with the pET28a(+) vector system in Escherichia coli BL21(DE3), and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni2+-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). TN0949 can hydrolyze o-nitrophenyl β-D-galactopyranoside at the optimum pH and temperature of 6.5 and 80 °C, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 °C, respectively. The Km values for the hydrolyses of o-nitrophenyl β-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH(3.0 to 7.0) after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 °C were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.

Characterization of a thermostable recombinant β-galactosidase from a thermophilic anaerobic bacterial consortium YTY-70

In the present study, a thermophilic anaerobic bacterial consortium YTY-70 that produced thermostable β-galactosidase was isolated from a hot spring in Yongtai (Fujian, China). Based on 16S rRNA sequences, we concluded that Caldicellulosiruptor was the predominant genus in the studied bacterial consortium. Subsequently, a thermostable β-galactosidase gene was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The recombinant enzyme exhibited a maximum activity at an optimum pH of 7.0 and an optimum temperature of 75 °C. It had a high activity across a broad pH range (between pH 5.0 and pH 9.0) and high thermal stability at temperatures up to 90 °C. The activity of the recombinant β-galactosidase was enhanced by TritonX-100, Tween-20 and 1 mmol/L 2-mercaptoethanol, while it was inhibited by ethylenediaminetetraacetic acid, sodium dodecyl sulphate, phenylmethylsulphonyl fluoride and 10 mmol/L 2-Me. The enzyme's catalytic function was enhanced by the following metal ions: Na+, K+, Mg2+, Ba2+, Ca2+, Fe2+, Zn2+, Mn2+, Al3+, Cu2+, Fe3+ and Li+. The thermostable β-galactosidase might be a promising candidate for use in food industries, particularly for the dairy industry and biosynthesis of galacto-oligosaccharides, due to its high thermostability and broad pH range.

A differentially conserved residue (Ile42) of GH42 β-galactosidase from Geobacillus stearothermophilus BgaB is involved in both catalysis and thermostability

The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable β-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 β-galactosidases, BgaB from Geobacillus stearothermophilus and A4-β-Gal from Thermus thermophilus A4. The A4-β-Gal has a catalytic triad (Glu312-Arg32-Glu35) with an extended hydrogen bond network that has not been observed in BgaB. In this study, we performed site-saturation mutagenesis of Ile42 in BgaB (equivalent to Glu312 in A4-β-Gal) to study the effects of different residues on thermostability, catalytic function, and the extended hydrogen bond network. Our experimental results suggest that substitution of Ile42 with polar AA enhanced the thermostability but decreased the catalytic efficiency of BgaB. Polar AA substitution for Ile42 simultaneously affected thermostability, catalytic efficiency, and the hydrogen bond network, suggesting that Ile42 is responsible for functional discrimination between members of the GH42 family. These observations could lead to a novel strategy for investigating the functional evolution of the GH42 β-galactosidases.

A novel thermostable β-galactosidase from Geobacillus kaustophilus HTA42

A novel thermostable β-galactosidase gene, designated as GkGal1A, from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli(E. coli). Based on the sequence analysis, GkGal1A belongs to the glycosyl hydrolase family 1 that was the first β-galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGal1A determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p-nitrophenyl-β-D-galactopyranoside at pH 7.8 and 70 °C and displayed high thermal stability. Divalent cations are prerequisite for the activity of GKGal1A, with the highest activity in the presence of Mn2+. Moreover, the three-dimensional structure of GkGal1A was modeled to speculate the structure of the catalytic residues and the reaction mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis.