Part:BBa_M45077
Heavy Metal Binding Protein (hmbp) gene
HMBP gene acquired from a genetic DNA library of activated-sludge microorganisms (accession number AB252419) and its deduced amino acid sequence.
Contents
Usage and Biology
hmbp is an important gene to be considered for the environmental biotechnology field that focuses on heavy metal removal from wastewater effluents. Currently, bio-adsorption is one of the techniques that are been extensively exploited, in which affinity and specificity of heavy metal binding are utilized. The advantages of HMBP as a binding protein for wastewater bioremediation is that hmbp was isolated from microorganisms that live in the same environment that the protein will be used. Thus, HMBPs derived from activated sludge would be stable and useful in water and wastewater treatment processes, because it is a fact that these HMBPs existed in activated sludge. There have been a number of studies with regard to the interaction of heavy metals and proteins, but the approach that HMBPs derived from environmental samples can be utilized as new materials for heavy metal adsorbents in water and wastewater treatments has not been pursued. The acquisition of HMBP from activated sludge could be the first step to establish new schemes for heavy metal removal from contaminated water. There will be several difficulties in establishing the functional protein-based technology for water and wastewater treatments, because such technology needs to produce and purify a large amount of HMBP and to test the durability performance. However, the application of functional biomaterials like HMBP might create a new horizon of research and development in the water and wastewater treatment engineering in the near future.
Characteristics
Studies show that HMBP might be a digestive product of a protein that has a high sequence similarity with DnaK involved in chaperon machinaries. The relative MW of the producted HMBP is reported to be about 30,000 and the MW from its aminoacid composition was 15,000.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 82
Functional Parameters
HMBP has only one histidine in its amino acid sequence. The binding constant of an average protein with a single histidyl residue reaches 4.5x10^3 M^-1, and the tight binding of soft metal ions such as copper ion to proteins can be achieved by multiple interactions between several histidyl residues and metal ion. However, HMBP has a large amount of other metal-coordinating amino acids, which are distributed over the tertiary structure of HMBP. The estimated tertiary structure also indicates that HMBP mainly consists of alpha helixes, in which residues are exposed to the outside. These residues of metal-coordinating amino acids projecting out to the water phase could play a significant role in forming several coordinating bonds with copper ion.
Experimental findings
The heavy-metal-binding ability of HMBP was evaluated with immobilized metal affinity chromatography. HMBP was trapped by nickel ion in the affinity column. HMBP in nickel ion-immobilized column was easily washed out by wash buffer injections with pH values of 6.0 and 5.0. On the other hand, HMBP was trapped by the copper ionimmobilized column as well, and HMBP binding to the copper ion in the affinity column could not be washed out at pH values of 6.0, 5.0, and 4.0. HMBP in the copper-immobilized column could not be eluted even at a pH value of 3.5 , in which the HMBP molecule would have a net positive charge due to its pI value of 4.2. EDTA-containing buffer was required to recover HMBP from the copper-immobilized column. These results mean that HMBP has a higher binding affinity to copper ion than to nickel ion. The order of affinity binding observed in this study partially conforms with the Irving-Williams series, which is the relative affinities of the first low divalent transition metals, Mn(II) < Co(II) < Ni(II) < Cu(II) < Zn(II). This series is the general trend in metal ion affinities observed for small-molecule chelators and also has been observed in interactions of metalbinding proteins. It is considered that HMBP can capture copper ion with several coordinating bonds, which overcome the simple electric repulsive force between HMBP and copper ion.
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