Indian Journal of Biochemistry & Biophysics
CODEN : IJBBBQ
VOLUME 39
ISSN : 0301-1208
NUMBER 4
AUGUST 2002
CONTENTS
Minireview
Mass spectrometry and protein sturucture
Kapil Maithal and K Muralidhar*
Applied Biocatalysis (Mini Symposium-in-Print)
About this Mini Symposium
Applied Biocatalysis: An Overview
Munishwar Nath Gupta* and Ipsita Roy
Papers
Covalent immobilization of cyclodextrin glycosyltransferase (CGTase) in
activated silica and Sepharose
M Teresa Martin, Miguel Alcalde, Francisco J Plou and Antonio Ballesteros*
205
218
220
229
Overexpression and protein folding of a chimeric ß-glucosidase
constructed from Agrobacterium tumefaciens and Cellvibrio gilvus
S P Singh*, J D Kim, S Machida and K Hayashi
235
Product conformation driven splicing of unprotected peptides by reverse proteolysis:
Influence of intrinsic and extrinsic factors
Sonati Srinivasulu and A Seetharama Acharya*
240
Depolymerization of starch and pectin using superporous matrix supported enzymes
Arvind Lali*, Kushal Manudhane, Nuzhat Motlekar and Priti Karandikar
Pseudomonas cepacia lipase-mediated transesterification reactions of
hydrocinnamates
K Priya, T Venugopal and Anju Chadha*
Synthesis and biochemical evaluation of benzyl propargyl ethers as
inhibitors of 5-lipoxygenase
N B Barhate, Madhava C Reddy, P Srinivas Reddy, R D Wakharkar and
P Reddanna*
253
259
264
Contd.
Immunoaffinity layering enhances the sensitivity of antigen detection on
nitrocellulose strips
Hina Jamil and Mohammed Saleemuddin*
274
Resolution of a complex ionic mixture of an apparently homogenous
protein preparation by preparative electrophoresis
Ashok K Dubey
279
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.205-216
Mass spectrometry and protein structure
Mini review
Kapil Maithal1 and K Muralidhar2*
Received 24 April 2002; accepted 6 June 2002
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.220-228
Applied Biocatalysis: An Overview
Munishwar N Gupta* & Ipsita Roy
Received 12 July 2002
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.229-234
Covalent immobilization of cyclodextrin
glucosyltransferase (CGTase) in activated silica and
Sepharose
M Teresa Martín, Miguel Alcalde, Francisco J Plou and Antonio Ballesteros*
Received 14 June 2002; accepted 19 June 2002
Cyclodextrin glucanotransferase is a non-Leloir glycosyltransferase that directly employs the free
energy of cleavage of starch to produce cyclodextrins. In presence of appropriate acceptors, this enzyme
synthesizes oligosaccharides containing (14) bonds. We have investigated the covalent
immobilization of CGTase onto different activated supports. Silica was aminated and further activated
with glutaraldehyde. The maximum amount of bound protein was about 4 mg CGTase per gram of
support; however, the catalytic efficiency of the immobilized enzyme was lower than 6%. Sepharose 4B
activated with cyanogen bromide (CNBr-activated Sepharose) and Sepharose 4B with a spacer arm of 1,6diaminohexane (EAH Sepharose) were also assayed. These gels react with the amino and carboxylic
groups of CGTase, respectively. With CNBr-activated Sepharose, a low percentage of enzyme was bound
to the support but with a significant catalytic efficiency (29%). A higher recovery of protein was obtained
with EAH Sepharose (62%), but only 2.4% of the initial activity was present in the immobilized
biocatalyst. The results were discussed in terms of CGTase structure and mechanism. In addition, the
solvent accessibility of amino or carboxylic groups, calculated using the NACCESS software, was
considered.
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.235-239
Overexpression and protein folding of a chimeric glucosidase constructed from Agrobacterium tumefaciens
and Cellvibrio gilvus
S P Singh*2, J D Kim@1, S Machida1 and K Hayashi1
Received 28 April 2002; revised and accepted 9 June 2002
In continuation of our investigation on structure and function relationship of -glucosidases from
mesophilic and thermophilic bacteria, we constructed a chimeric gene by shuffling 17% length in C
terminal region of ß-glucosidase of Agrobacterium tumefaciens with the corresponding homologous
region of Cellvibrio gilvus -glucosidase. The chimeric gene was overexpressed in E. coli BL21 (DE3)
using pET vector. However, nearly all of the -glucosidase produced was trapped into inclusion bodies in
catalytically non-functional state. Attempts were made to solubilize the overexpressed protein by coexpression with molecular chaperone, GroEL/ES, in vivo. The molecular chaperone assisted protein
folding that had earlier yielded encouraging results, did not improve the solubilization in the present case
with a chimeric -glucosidase. Further, we explored protein renaturation under in vitro conditions using
various dialysis strategies. Dialysis, rapid dilution and a newly devised method of folding immobilized
proteins yielded active enzyme. The usefulness of the in vitro folding methods to obtain functional
enzymes from overproduced but non-functional proteins has been discussed.
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.240-252
Product conformation driven splicing of unprotected
peptides by reverse proteolysis: Influence of intrinsic and
extrinsic factors
Sonati Srinivasulu1 and A Seetharama Acharya1,2 *
Received 11 June 2002; revised and accepted 5 July 2002
The structural motif of ‘product conformation driven V8 protease catalyzed ligation reaction’ can be
represented by FRI-EALER-FRII. The relative roles of the flanking regions (FRI and FRII) and of
splicedon, the central penta-peptide, on the thermodynamic stability of the ‘conformational trap’ of the
product has been now evaluated as a function of co-solvent concentration. The studies have established
that the thermodynamic stability of the conformational trap of 17-40des23-26 with four different splicedons
(EALER, EALEV, EYGER, or EGAER) that differ in the intrinsic -helical potential of their amino acid
residues and/or ability to generate i, i+4 side chain interaction is a direct correlate of the n-propanol
induced -helical conformation of the product. On the other hand, when the product is defined by only
splicedon EALER, and the flanking regions are disitinct; no correlation could be drawn between the
stability of the trap and solvent induced - helical conformation, even though these generally give an
equilibrium yield of 45% in 30% n-propanol and is not influenced by an increased propanol concentration.
However, when the splicedon EALER with given FRI and FRII region develops a ‘conformational trap’ of
a lower stability in 30% propanol as seen with 18-25(A22)-EALER-31-39, the stability increases in 60% n-
propanol, without significant increase in the - helical conformation. Though, primary structure of
RNAse1-20, could be presented as RNAse1-5-AKFER- RNAse11–20, and -helical conformation is induced
to this peptide both in 30 and 60% propanol, splicedon AKFER by itself does not develop the
‘conformational trap’ of RNAse1-20. The splicedon AKFER of RNAse1- 20 fails to develop the
‘conformational trap’, due to an intrinsic inhibitory potential of its FR II region, RNAse11-20; replacing
RNAse11-20 with 32-40 enables the splicedon AFKER to generate the ‘conformational trap’. The studies
presented here have demonstrated the primary role of flanking regions in establishing the amount of the
solvent induced -helical conformation and that of the splicedon in dictating the thermodynamic stability
of its ‘conformational trap’ of the products, nonetheless one influences the other to some degree. We
suggest that the stability of the ‘conformational trap’ of the product reflects the ability of the splicedon to
‘recruit’ the product conformation to protect the spliced peptide bond, i.e. to reduce the helix-coil
transition of the spliced region which in turn imparts a degree of resistance to the spliced peptide bond.
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.235-258
Depolymerization of starch and pectin using superporous
matrix supported enzymes
Arvind Lali*, Kushal Manudhane, Nuzhat Motlekar and Priti Karandikar
Received 23 June 2002; revised and accepted 28 June 2002
Immobilized enzyme catalyzed biotransformations involving macromolecular substrates and/or
products are greatly retarded due to slow diffusion of large substrate molecules in and out of the typical
enzyme supports. Slow diffusion of macromolecules into the matrix pores can be speeded up by use of
macroporous supports as enzyme carriers. Depolymerization reactions of polysaccharides like starch,
pectin, and dextran to their respective low molecular weight products are some of the reactions that can
benefit from use of such superporous matrices. In the present work, an indigenously prepared rigid crosslinked cellulose matrix (called CELBEADS) has been used as support for immobilizing alpha amylase
(1,4--D-glucan glucanohydrolase, EC 3.2.1.1.) and pectinase (endo-PG: poly(1,4--galactouronide)
glycanohydrolase, EC 3.2.1.15). The immobilized enzymes were used for starch and pectin hydrolysis
respectively, in batch, packed bed and expanded bed modes. The macroporosity of CELBEADS was
found to permit through-flow and easy diffusion of substrates pectin and starch to enzyme sites in the
porous supports and gave reaction rates comparable to the rates obtained using soluble enzymes.
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.259-263
Pseudomonas cepacia lipase - mediated transesterification
reactions of hydrocinnamates
K Priya, T Venugopal and Anju Chadha*
Received 10 May 2002; revised and accepted 11 June 2002
Use of lipase from Pseudomonas cepacia in transesterifcation reactions of ethyl hydrocinnamate with
different alcohols has been examined. Among the alcohols tested, viz., n-butanol, iso-amyl alcohol,
benzyl alcohol, n-octanol and 1-phenylethanol, only n-butanol yielded the transesterified product. Among
the solvents tested, viz., n-heptane, n-hexane, cyclohexane, toluene, diisopropylether and n-butanol, the
initial rate of transesterification proceeded in the order cyclohexane > n-heptane > n-hexane >
diisopropylether > n-butanol > toluene. Using hexane as the solvent and a substrate to enzyme ratio of 1:5,
the substrate to alcohol ratio was varied to maximize the yield. n-Butyl hydrocinnamate was obtained in
92% yield in 48 hr by employing a 1:1 (wt/wt) ratio of ethyl hydrocinnamate to lipase and a 1:5 (vol/vol)
ratio of ethyl hydrocinnamate to n-butanol in hexane.
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.264-273
Synthesis and biochemical evaluation of benzyl propargyl
ethers as inhibitors of
5-lipoxygenase
N B Barhatea, Madhava C Reddy, P Srinivas Reddy, R D Wakharkar a and P Reddanna*
Received 18 May 2002; revised and accepted 18 June 2002
A series of benzyl propargyl ethers were synthesized and tested as inhibitors of 5-lipoxygenase, the
key enzyme involved in leukotriene biosynthesis. Among these, optimum activity was displayed by 1-(2heptynyloxymethyl) benzene 12 (IC50 1.2 M). Addition of carboxyl group at the end of the alkyl side
chain attached to the acetylenic group abolished the inhibition. Selective reduction of the acetylenic group
to cis or trans double bond reduced the inhibitory potential, the cis isomer 24 showing more than 20-fold
higher inhibition than the trans isomer 25. Introduction of sulphur in place of oxygen in the alkyl side
chain attached to the (carboxyalkyl) benzyl group also reduced the inhibition. The IC 50 value of 12,
towards rabbit reticulocyte 15-LOX is > 50 fold higher than that of 5-LOX. These results indicate that
compound 12 is a specific inhibitor of 5-LOX.
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.274-278
Immunoaffinity layering enhances the sensitivity of antigen
detection on nitrocellulose strips
Hina Jamil1 and Mohammed Saleemuddin*1, 2
Received 17 April 2002; revised and accepted 17 June 2002
A simple strategy to remarkably increase the sensitivity of detection of antigens applied as dot or
western blot on nitrocellulose membrane using human serum albumin as model antigen has been
described. This involves subjecting the antigen bearing nitrocellulose strips to multiple incubation cycles
with primary antibody and enzyme conjugated secondary antibody prior to staining for enzyme activity.
The sensitivity of detection could be increased up to a thousand fold after three incubation cycles.
Aggregation of human serum albumin could be detected by the multiple incubation procedure at very low
protein concentration after electrophoresis and transfer onto nitrocellulose.
Indian Journal of Biochemistry & Biophysics
Vol. 39, August 2002, pp.279-283
Resolution of a complex ionic mixture of an apparently
homogenous protein preparation by preparative
electrophoresis
Ashok K Dubey*
Received 3 May 2002; revised and accepted 30 May 2002
Preparations of recombinant envelope glycoprotein E2 of hepatitis C virus (r-HCV E2), found to be
homogeneous by N-terminal amino acid sequencing and mass spectrometry, resolved into multiple ionic
species (isoforms) when analysed by isoelectric focusing (IEF) gel electrophoresis in the pI range of 3-10.
These isoforms possessed pI values in the range of 4.5-8.2. The major isoform with pI value of
approximately 7.1 was separated from the rest of them by employing a method developed on Gradiflow
BF 200, a device based on preparative electrophoresis. This isoform was adjudged to be homogenous by
IEF and by native polyacrylamide gel electrophoresis (PAGE).