Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 216

Histidine-rich or polyhistidine affinity tails can be associated with proteins
and enzymes by fusing the coding sequence of the former with those of the latter
[147,148]. Such fusion proteins could be immobilized by taking advantage of the
specific binding ability of the affinity peptide to appropriate IMA supports.
Ljunquist et al. [149] observed that -galactosidase fusion protein bearing a

histidine-rich peptide extension at N -terminus was bound to Zn 2+ -IDA adsor-
bent and retained significant enzyme activity. The activity was however lost
once the enzyme was eluted,which according to the authors,was the result of
inhibition of the enzyme by Zn 2+ leaking from the IDA supports. In order to
overcome the problems of metal ion bleeding,Piesecki et al. [150] immobi-
lized

b

-galactosidase fusion protein comprising of six histidine residues at the
N -terminus to Ni 2+ -nitricotriacetic acid (NTA) adsorbent that binds metal ion
far more strongly than the IDA adsorbent [151]. NTA groups form tetracoordi-
nated complexes with metal ions leaving two free coordination sites,while IDA
groups form tricoordinate complexes leaving three coordination sites [138]. The
association between metal ion and support may thus be stronger in case of NTA
substituent,while that between protein and metal loaded support is stronger in
case of supports having IDA [81].
Carlsson et al. [152] in a more recent study determined the activities of several
enzymes with genetically attached polyhistidine tails bound to metal chelate
supports. These included lactate dehydrogenase (his) 4 ,galactose dehydrogenase
(his) 5 ,

b

-glucouronidase (his) 4 and the complex prepared using protein A
(his) 5 and horse radish peroxidase labelled immunoglobulin (protein A (his) 5
IgG HRP). All enzymes exhibited good activity in the immobilized state but pro-
tein A (his) 5 IgG HRP was most active and exhibited essentially same activity as
the soluble complex both on Cu 2+

b

and Zn 2+ supports. Activities of galactose

dehydrogenase(his) 5 and -glucouronidase (his) 4 were high if Zn 2+ instead

of Cu 2+ was used on the chelating material. Enzymes however showed a lower
tendency to bleed from the chelated copper supports. All immobilized enzymes
exhibited good storage stability which was at least in the same range as that of
the respective soluble enzyme.
A chemical procedure for the enhancement of the surface histidine content of
glycoenzymes in order to improve their affinity for metal chelate supports is also
available [153]. The method involves oxidation of the carbohydrate moiety of
the glycoprotein with periodate followed by covalent modification with histi-
dine. Using the strategy it was possible to increase remarkably the affinity of
Pencillium chrysogenum glucose oxidase for metal ion chelated supports and
confer affinity to horse radish peroxidase that does not bind to the support in the
native form. The histidine-enriched enzymes exhibited high activity,remark-
able stability and could be used effectively in the analysis of the glucose.
Several proteins and enzymes have innate affinity in their native state for the
metal chelate supports due to the presence of one or more surface histidines
(Table 3). Coulet et al. [154] immobilized lactate dehydrogenase,malate dehy-
drogenase and alkaline phosphatase on Co 2+ ,Zn 2+ ,Cu 2+ -chelate Sepharose. All
the enzymes were active in the immobilized state although the retention of the
specific activity was quite low. Among the enzymes used,alkaline phosphatase
retained higher activity and among the various metal chelates maximum activi-

b