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Extremophile Protein Database
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Alkaliphilic microorganisms are capable of growing under alkaline (pH roughly 8.5-11) environments. Alkaliphilic microorganisms (or alkaliphiles) belong to all the three domains: eukaryote, bacteria, and archaea. The cell surface may play a key role in maintaining intracellular pH homeostasis close to neutrality (Horikoshi 1999). Alkaliphiles have made a great impact in industrial applications such as in detergent formulations, foodstuffs, chemicals and pharmaceuticals. The biological detergents contain alkalistable enzymes, such as proteases, lipases, cellulases, that have been produced from alkaliphiles. The production of cyclodextrin by alkaline cyclomaltodextrin glucanotransferase is an another important industrial application. The economic production of cyclodextrin were exploited for the foodstuffs and pharmaceuticals synthesis (Horikoshi 1999).

Factors responsible for Alkalistability

Genomic adaptations

Like other extremophiles, the identification of alkaliphilic genomes repertoire are also less reported. The genome search with the key word "alkaliphilic" in NCBI genome search results in a total of 13 hits for alkaliphiles. Corresponding to the acidophiles, alkaliphilic genomes are adapted to maintained pH homeostasis. In their genome, several open reading frames for Na+ /H+ antiporters responsible for pH homeostasis in alkaliphiles have been characterized (Horikoshi 1999). Alkylphosphonate ABC transporter genes coding for two permeases, one phosphonate-binding protein and one ATP-binding protein, are the most frequent class of protein coding gene expressed in alkaliphiles. These transporters couple the hydrolysis of ATP to solute transport (Takami et al., 1999). Recently, the complete genome of Bacillus subtilis and Bacillus halodurans C-125 has been sequenced. Genes responsible for the alkaliphily of B. halodurans C-125 and Bacillus firmus OF4 have been analyzed (Takami and Horikoshi 2000). The tupA gene was identified in the B. halodurans genome, which is responsible for the synthesis of teichuronopeptide, a major structural component in the cell wall important for maintaining pH homeostasis (Takami and Horikoshi 2000).

Proteomic adaptations

The comparative studies along with experimental and theoretical analysis have led to three main conclusions (Siddiqui and Thomas 2008). First, the pKa modulation of a catalytic residue toward higher pH is responsible for alkaline protein stability. This is achieved through modification of the hydrogen bonds and reduction in solvent exposure of the catalytic residue. For Instance, the two thermoalkaliphilic xylanases, designated XylA and XylB from Bacillus sp. AR-009 have mixed thermophilic and alkaliphilic behaviours. Both enzymes are stable in a broad pH range and shows good stability when incubated at 60° C and 65deg; C in pH 8 and pH 9 buffers respectively (Gessesse 1998). Second, an increase in the surface exposure of acidic residues with respect to basic residues changes the net charge of the molecule toward negative. GH10 alkaline xylanase BSX from Bacillus sp. NG-27 and alkaline phosphosereine aminotransferase from Bacillus alcalophilus show such a trend (Siddiqui and Thomas 2008). Third, the alkaliphiles are adapted to an increase in the surface exposure of acidic residues with respect to basic residues changes the net charge of the molecule towards negative. The gain of glutamate plus arginine residues and the loss of aspartate plus lysine residues are key players in alkaline adaptation of proteins. Moreover, during the adaptation process it was observed that smaller hydrophobic residues were gained and larger ones lost when enzymes from alkalophiles and non-alkalophiles were compared (Siddiqui and Thomas 2008). Modification of the proteome by increasing the fraction of acidic amino acids and reducing the protein hydrophobicity for alkaline stability has been observed in the haloalkaliphilic archaeon Natronomonas pharaonis (Horikoshi, 1998). It was also suggested to mutate Asn residues to improve stability at extreme alkaline conditions (Palmer et al. 2008). Since deamidation of Asn residues is a spontaneous and non-enzymatic reaction which can occur in alkaline conditions. In alkaline condition, it was also established that Asn deamidate and leading to destabilization of the protein structure (Robinson 2002).

Further Reading

Gessesse A (1998) Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillussp Applied and Environmental Microbiology 64:3533-3535.

Horikoshi K (1999) Alkaliphiles: some applications of their products for biotechnology Microbiology and molecular biology reviews 63:735-750.

Palmer B, Angus K, Taylor L, Warwicker J, Derrick JP (2008) Design of stability at extreme alkaline pH in streptococcal protein G Journal of biotechnology 134:222-230.

Robinson NE (2002) Protein deamidation Proceedings of the National Academy of Sciences 99:5283-5288.

Siddiqui KS, Thomas T (2008) Protein adaptation in extremophiles, Nova Publishers.

Takami H, Horikoshi K (2000) Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view Extremophiles 4:99-108.