Purification, characterization, and comparison of anα-glucosidase (endo-oligo-1, 4-glucosidase) from the mesophileBacillus subtilis and the obligate thermophileBacillus caldolyticus

Krohn, Bradley M.; Lindsay, James

doi:10.1007/bf02105389

Current Microbiology

1991

DOI: 10.1007/bf02105389

|View full text |Cite

Purification, characterization, and comparison of anα-glucosidase (endo-oligo-1, 4-glucosidase) from the mesophileBacillus subtilis and the obligate thermophileBacillus caldolyticus

Bradley M. Krohn

James Lindsay

Help me understand this report

Search citation statements

Order By: Relevance

Paper Sections

Select...

Citation Types

Supporting

Mentioning

Contrasting

Year Published

1991

2000

Publication Types

Select...

Article5

Relationship

Self Cite0

Independent5

Authors

Journals

Cited by 7 publications

(1 citation statement)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Most known α-glucosidases do not have an essential need of metal cations for activity. On the contrary, moderate to severe inhibition by Mn 2ϩ and Co 2ϩ has been reported for some α-glucosidases, i.e., the Thermococcus species strain AN1 enzyme and some Bacillus enzymes (Suzuki et al 1992;Piller et al 1996;Krohn and Lindsay 1991). The precise role for Mn 2ϩ for enzyme activity of the glycosyl hydrolase family 4 enzymes is uncertain.…”

mentioning

confidence: 98%

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

et al. 2000

View full text Add to dashboard Cite

The gene for the alpha-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hydrolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritima alpha-glucosidase has the unusual property of requiring NAD+ and Mn2+ for activity. Co2+ and Ni2+ also activated AglA, albeit less efficiently than Mn2+. T. maritima AglA represents the first example of a maltodextrin-degrading alpha-glucosidase with NAD+ and Mn2+ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or beta-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD+ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of D-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90 degrees C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50 degrees C and 70 degrees C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h.

show abstract

mentioning

confidence: 98%

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

et al. 2000

View full text Add to dashboard Cite

show abstract

Purification and Characterization of an Oligo‐1,6‐glucosidase from the Caldoactive Thermophile Bacillus caldotenax

1997

View full text Add to dashboard Cite

An oligo‐1,6‐glucosidase (dextrin 6‐α‐D‐glucanhydrolase, EC 3.2.1.10) of the caldoactive thermophile Bacillus caldotenax KP 1213 (YT‐G, DSM406) was purified to homogeneity. Its relative molecular weight, Stokes radius, sedimentation coefficient at 20°C in water, molar absorption coefficient at 280nm and pH 6.8, and isoelectric point were estimated to be 64,000, 3.3nm, 4.8S, 122,000M−1cm−1, and 4.9, respectively. The enzyme N‐terminal sequence of twenty residues was determined to be Met1‐Glu‐Trp‐Ala‐Trp‐Lys‐Glu‐Ala‐Val‐Val‐Tyr‐Gln‐Ile‐Tyr‐Pro‐Arg‐Met‐Phe‐Tyr20. The enzyme shared its antigenic groups in part with the homologue from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile). The B. caldotenax enzyme was most active at 70°C and at pH 6.0–6.8. The best substrate for the enzyme was isomaltotriose of naturally‐occurring oligo‐ and polysaccharides tested. The enzyme half‐life at pH 6.8 was 10min at 75°C. The enzyme (Pro, 5.93mol%) fairly matched with a positive relationship between the thermostability of its six bacillary counterparts and their proline mol% contents. This relationship has been found to hold for sixteen bacterial enzymes from other four different groups (α‐glucosidases, pullulanases and 3‐isopropylmalate dehydrogenases).

show abstract

Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

Krohn

Lindsay

1993

Current Microbiology

View full text Add to dashboard Cite

The cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17 was cloned on separate PstI, BamHI, and EcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host, Escherichia coli DH5 alpha. High level constitutive expression of the gene product was also detrimental to the E. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kb EcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the host B. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in the B. subtilis host; however, expression was at a low level. Subcloning of the 3-kb EcoRI fragment into pUC18 and transformation into E. coli XL1-Blue (F' lacIq) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from the Bacillus temperature phage SPO2 promoter of pPL708 may increase expression of this gene.

show abstract

scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.

Contact Info

customersupport@researchsolutions.com

10624 S. Eastern Ave., Ste. A-614

Henderson, NV 89052, USA

This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.

Blog Terms and Conditions API Terms Privacy Policy Contact Cookie Preferences Do Not Sell or Share My Personal Information

Made with 💙 for researchers

Part of the Research Solutions Family.

Purification, characterization, and comparison of anα-glucosidase (endo-oligo-1, 4-glucosidase) from the mesophileBacillus subtilis and the obligate thermophileBacillus caldolyticus

Cited by 7 publications

References 22 publications

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

Purification and Characterization of an Oligo‐1,6‐glucosidase from the Caldoactive Thermophile Bacillus caldotenax

Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

Contact Info

Product

Resources

About