Indian Journal of Biochemistry & Biophysics
CODEN : IJBBBQ ISSN : 0301-1208
|
VOLUME 39 |
NUMBER 4 |
AUGUST 2002 |
CONTENTS
|
Minireview |
|
|
Mass
spectrometry and protein sturucture |
205 |
|
|
|
|
|
|
|
Applied Biocatalysis (Mini
Symposium-in-Print) |
|
|
About
this Mini Symposium |
218 |
|
|
|
|
Applied
Biocatalysis: An Overview |
220 |
|
|
|
|
|
|
|
Papers |
|
|
Covalent immobilization of cyclodextrin
glycosyltransferase (CGTase) in activated silica and Sepharose |
229 |
|
M Teresa Martin, Miguel Alcalde,
Francisco J Plou and Antonio Ballesteros* |
|
|
|
|
|
Overexpression and protein
folding of a chimeric ß-glucosidase constructed
from Agrobacterium tumefaciens and Cellvibrio gilvus |
235 |
|
|
|
|
|
|
|
Product conformation driven splicing of unprotected
peptides by reverse proteolysis: Influence of intrinsic and extrinsic factors |
240 |
|
|
|
|
|
|
|
Depolymerization
of starch and pectin using superporous matrix supported enzymes |
253 |
|
Arvind Lali*, Kushal
Manudhane, Nuzhat Motlekar and Priti Karandikar |
|
|
|
|
|
Pseudomonas cepacia lipase-mediated transesterification reactions of hydrocinnamates |
259 |
|
|
|
|
|
|
|
Synthesis and biochemical evaluation of benzyl
propargyl ethers as inhibitors of 5-lipoxygenase |
264 |
N B Barhate, Madhava
C Reddy, P Srinivas Reddy, R D Wakharkar and
P Reddanna*
|
|
|
|
|
|
|
|
|
|
Contd. |
|
Immunoaffinity layering
enhances the sensitivity of antigen detection on nitrocellulose
strips |
274 |
|
|
|
|
|
|
|
Resolution of a complex ionic mixture of an
apparently homogenous protein preparation by preparative
electrophoresis |
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 a(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 1,6-diaminohexane (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 b-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 b-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 b-glucosidase. The chimeric gene was
overexpressed in E. coli BL21 (DE3) using pET vector. However,
nearly all of the b-glucosidase produced was trapped into inclusion bodies in catalytically
non-functional state. Attempts were made to solubilize the overexpressed
protein by co-expression 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 b-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 a17-40des23-26 with four different splicedons (EALER, EALEV, EYGER, or EGAER) that differ in the intrinsic a-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 a-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 a- 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 b18-25(A22)-EALER-b31-39, the stability increases in 60% n-propanol, without significant increase in the a- helical conformation. Though, primary structure of RNAse1-20, could be presented as RNAse1-5-AKFER- RNAse11–20, and a-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 FRII region, RNAse11-20; replacing RNAse11-20 with a32-40 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 a-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 cross-linked cellulose matrix (called CELBEADS) has been used as support for immobilizing alpha amylase (1,4-a-d-glucan glucanohydrolase, EC 3.2.1.1.) and pectinase (endo-PG: poly(1,4-a-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 Wakharkara 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-(2-heptynyloxymethyl) benzene 12 (IC50 1.2 mM). 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 IC50 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).