Extracellular secretion in Bacillus subtilis of a cytoplasmic thermostable β-galactosidase from Geobacillus stearothermophilus

Xia, Yu; Zhao, Jianxin; Chen, H.; Liu, X.; Wang, Y.; Tian, Fang; Zhang, H.P.; Zhang, H.; Chen, Wei

doi:10.3168/jds.2009-2864

Cited by 25 publications

(22 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The β-galactosidase activity was determined as previously described (Xia et al, 2010) with some modifications. The 5.0-mL substrate solution (ONPG, 0.5 mg/ mL, pH 7.0) was mixed with 0.9 mL of PBS (pH 7.0) and the mixture was kept in water bath at the optimum temperature of the enzyme for 30 min.…”

Section: Enzymatic Activity Assaymentioning

confidence: 99%

Purification, characterization, and gene cloning of a new cold-adapted β-galactosidase from Erwinia sp. E602 isolated in northeast China

Xia

Mao

et al. 2018

Journal of Dairy Science

View full text Add to dashboard Cite

β-Galactosidases are widely used in industry for elimination of lactose from milk products. A new β-galactosidase was obtained from bacterial strain Erwinia sp. E602, newly isolated in northeast China. The enzyme was purified with the methods of ammonium sulfate fractionation, ion exchange, and gel filtration chromatography for further study of the enzymatic characteristics. The purified enzyme had a molecular weight of near 110 kDa. The optimum reaction temperature and pH of this enzyme was determined to be 40°C and 7.0, respectively, indicating that this enzyme was a mesophilic neutral β-galactosidase. Furthermore, the enzyme retained near 10% of the activity at 0°C, which also suggested its cold-adapted property. Kinetics of the β-galactosidase was studied, and the K (Michaelis constant) and V (maximum enzymatic reaction rate) of this enzyme were 0.21 mmol/L and 263.16 µmol/mg per minute, respectively. The effects of metal ions on the enzymatic activity and the lactose hydrolysis efficiency in milk, as well as its trans-glycosylation activity, were studied in this work. The β-galactosidase coding gene was cloned to be a 3-kb length fragment, which shared at most 81% of identity with the published sequences in NCBI Blast database (https://blast.ncbi.nlm.nih.gov). Results in this work suggested it is a new β-galactosidase and it has potential to be used in dairy and food processing.

show abstract

Section: Enzymatic Activity Assaymentioning

confidence: 99%

Purification, characterization, and gene cloning of a new cold-adapted β-galactosidase from Erwinia sp. E602 isolated in northeast China

Xia

Mao

et al. 2018

Journal of Dairy Science

View full text Add to dashboard Cite

show abstract

“…Bacterial β-galactosidases have also been cloned and expressed in Bacillus subtilis (Chen et al, 2008;Xia et al, 2010). The thermostable β-galactosidase from Bacillus stearothermophilus was expressed in B. subtilis to overcome the lower enzymatic half-life at 60°C (just 0.5 h) presented by the homologous recombinant enzyme previously expressed in E. coli (Chen et al, 2008).…”

Section: Recombinant Expression Of Bacterial β-Galactosidasesmentioning

confidence: 99%

Recombinant microbial systems for improved β-galactosidase production and biotechnological applications

Oliveira

Guimarães

Domingues

2011

Biotechnology Advances

153

105

View full text Add to dashboard Cite

β-Galactosidases (EC 3.2.1.23) constitute a large family of proteins that are known to catalyze both hydrolytic and transgalactosylation reactions. The hydrolytic activity has been applied in the food industry for decades for reducing the lactose content in milk, while the transgalactosylation activity has been used to synthesize galacto-oligosaccharides and galactose containing chemicals in recent years. The main focus of this review is on the expression and production of Aspergillus niger, Kluyveromyces lactis and bacterial β-galactosidases in different microbial hosts. Furthermore, emphasis is given on the reported applications of the recombinant enzymes. Current developments on novel β-galactosidases, derived from newly identified microbial sources or by protein engineering means, together with the use of efficient recombinant microbial production systems are converting this enzyme into a relevant synthetic tool. Thermostable β-galactosidases (cold-adapted or thermophilic) in addition to the growing market for functional foods will likely redouble its industrial interest.

show abstract

“…The activities of BgaB variants were determined as described by Gist-Brocades (Stellmach, 1988); the incubation temperature of the enzyme was adjusted to 55°C (Xia et al, 2010). One unit of enzyme activity was defined as the amount of enzyme hydrolyzing 1 μmol of substrate (oNPG) per minute.…”

Section: Assay Of Galactosidase Activitymentioning

confidence: 99%

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Dong

Liu

Chen

et al. 2011

Journal of Dairy Science

Self Cite

View full text Add to dashboard Cite

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (k(cat)/K(m)). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.

show abstract

Extracellular secretion in Bacillus subtilis of a cytoplasmic thermostable β-galactosidase from Geobacillus stearothermophilus

Cited by 25 publications

References 27 publications

Purification, characterization, and gene cloning of a new cold-adapted β-galactosidase from Erwinia sp. E602 isolated in northeast China

Purification, characterization, and gene cloning of a new cold-adapted β-galactosidase from Erwinia sp. E602 isolated in northeast China

Recombinant microbial systems for improved β-galactosidase production and biotechnological applications

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Contact Info

Product

Resources

About