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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0266

PubMed

23320757, BMC Biotechnol. 2013 Jan 16;13:3. doi: 10.1186/1472-6750-13-3.

Characterization method

enzyme activity assay

Genomic accession number

NC_012028.1

Nucelotide position range

114293-135268

Substrate

lactose

Loci

HLAC_RS14125-HLAC_RS14200

Species

Halorubrum lacusprofundi/2247

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- HLAC_RS14125 WP_009486633.1 0 - 1167 (+) NC_012028.1:114293-115460 -
- HLAC_RS18870 WP_009486645.1 1281 - 1362 (-) NC_012028.1:115574-115655 -
- HLAC_RS14135 WP_012659234.1 2964 - 4137 (+) NC_012028.1:117257-118430 -
eda HLAC_RS14140 WP_012659236.1 5029 - 5668 (-) NC_012028.1:119322-119961 -
- HLAC_RS14145 WP_012659237.1 5728 - 6475 (-) NC_012028.1:120021-120768 -
- HLAC_RS14150 WP_049933796.1 6799 - 8158 (+) NC_012028.1:121092-122451 -
- HLAC_RS14155 WP_012659239.1 8176 - 9247 (+) NC_012028.1:122469-123540 -
- HLAC_RS14160 WP_012659240.1 9246 - 10083 (+) NC_012028.1:123539-124376 -
- HLAC_RS14165 WP_012659241.1 10085 - 11207 (+) NC_012028.1:124378-125500 -
dgoD HLAC_RS14170 WP_012659242.1 11213 - 12362 (+) NC_012028.1:125506-126655 4.2.1.6
- HLAC_RS14175 WP_049933798.1 12402 - 13257 (+) NC_012028.1:126695-127550 -
- HLAC_RS14180 WP_012659244.1 13304 - 15407 (-) NC_012028.1:127597-129700 -
- HLAC_RS14185 WP_012659245.1 15536 - 17576 (+) NC_012028.1:129829-131869 -
- HLAC_RS14190 WP_012659246.1 17706 - 18663 (+) NC_012028.1:131999-132956 -
- HLAC_RS14195 WP_012659247.1 19273 - 19705 (+) NC_012028.1:133566-133998 -
- HLAC_RS14200 WP_012659248.1 19701 - 20976 (+) NC_012028.1:133994-135269 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 1 - 1167 (+) CDS No
- 2965 - 4137 (+) CDS No
eda 5030 - 5668 (-) CDS No
- 5729 - 6475 (-) TF: DBD-Pfam|HTH_5,DBD-SUPERFAMILY|0040266 No
- 6800 - 8158 (+) TC: gnl|TC-DB|P29822|3.A.1.1.4 Yes
- 8177 - 9247 (+) TC: gnl|TC-DB|P29823|3.A.1.1.4 Yes
- 9247 - 10083 (+) TC: gnl|TC-DB|Q9WYR1|3.A.1.1.39 Yes
- 10086 - 11207 (+) TC: gnl|TC-DB|Q8TZQ3|3.A.1.1.16 Yes
dgoD 11214 - 12362 (+) other Yes
- 12403 - 13257 (+) other Yes
- 13305 - 15407 (-) CAZyme: GH42 Yes
- 15537 - 17576 (+) CAZyme: GH36 Yes
- 17707 - 18663 (+) STP: STP|PfkB No
- 19274 - 19705 (+) CDS No
- 19702 - 20976 (+) CDS No

PUL ID

PUL0266

PubMed

23320757, BMC Biotechnol. 2013 Jan 16;13:3. doi: 10.1186/1472-6750-13-3.

Title

Cloning, overexpression, purification, and characterization of a polyextremophilic beta-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi.

Author

Karan R, Capes MD, DasSarma P, DasSarma S

Abstract

BACKGROUND: Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures. RESULTS: Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 beta-galactosidase gene, named bga. In order to study the biochemical properties of the beta-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2) gene promoter. The recombinant beta-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (-5 to 60 degrees C) with an optimum of 50 degrees C, and 10% of its maximum activity at 4 degrees C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic beta-galactosidase was also stable and active in 10-20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol. CONCLUSION: The H. lacusprofundi beta-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is successful in a perennially cold, hypersaline environment, with relevance to astrobiology.