4. DISCUSSION

Studies employing the xerophytic plant species Opuntia vulgaris and Cereus

pterogonus have indicated the existence of multiple forms of the endoglucanase

activity in them. They could be categorised as temperature isoforms (30, 50, 70 and

90°C) and pH isofom (3, 4.515.5,7 & 9). Although the pH isoforms could only be

broadly differentiated as acidic and the neutral forms, the temperature isoforms were

distinctly identifiable as four different endoglucanase activities, each having their

characteristic temperature optimum. However, among these four thermoforms

identifiable in each species, only two forms were to be considered as thermophilic as

per the definition. These were the T70 and Tqo isoforms of both species. Having

identified the thermophilic isoforms in the above species, attempts were made to

determine their themostability over a range of temperature upto a maximum of

100°C, as also their propensity to denaturation through this temperature range. A

comparative determination of temperature profile of the endoglucanase activity in

the leaves of the mesophilic Ixora species when used as a control, indicated the

existence of temperature optimum in this species at 30 "C and 50 'C only.

Karl-Erik Eriksson and Bert Penerson, 1975; Poonsuk Prasertsan and Horst

W. Doelle 1986, serve as a important evidence for this work, Karl-Erik Eriksson

have characterised five endoglucanases from Sporolrichum pulverulenfum

(Chrysosporium lignorum) with different molecular weights and Poonsuk Prasertsan

have characterized six major endoglucanases from Cellulomonas species in his

studies. Krystyna Zoltowska (2001) had reported the existence of two isofoms of

amylase from the intestine and the muscle of Ascaris Suum (Nematoda). The

intestinal amylase showed two optimum activities, at 40°C and 50°C. The muscle

amylase also exhibited two optimum activities, at 30°C and 50°C. Nabi and

Srikumar (2003) reported the existence of temperature stable amylase isoforms from

Opunria vulgaris. The plant amylase showed two optimum activities at 50°C and

90°C. The existence of two forms of temperature stable enzyme in these species was

therefore considered as due to the different thermal sensitiveness. Ravikumar et al.,

(2007) reported the existence of the T60 and Tso isoforms in Cereus pterogonus

versus the existence of T40. T70 and T90 isoforms in opuntia vulgaris .Maloney et al.,

(1985) reported an endoglucanase of (35 kDa) from themophilic fungi Talaromyces

emersoniiare, with optimal activity between 75 to 80°C at pH 5.0 to 5.8. Susumu

Ando et al., (2002) reported a thermostable endoglucanase homolog from the

hyperthermophilic archaeon Pyrococcus horikoshii expressed in Escherichia coli,

which can withstand temperature of upto 97 "C.

Several cellulases from plants (Byme et a]., 1975), fungi (Macarron et a].,

1993 and Monti et al., 1991) or bacteria Sheweita et al., 1996 and Cavicchioli, 1991

show optimal hydrolytic activities between pH 4.0 and 6.0. Yan-Hong LI et al., 2005

reported the presence of a 27 kDa endoglucanase EG27 which is stable in a broad

range of pH 3.0 - 11 .O, showed unusual, acidophilic and alkaliphilic featwe. Kelvin

Eckert and Erwin Schneider, 2003 described a recombinant endoglucanase

(CelBtmc) exhibiting pH optima at 4 but still displayed 50% of its activity even at

pH 3 and 5. Bingze Xu et al., 2000 showed in blue mussel that, maximum activity

was obtained at pH 4.6 with a comparatively rapid decrease in activity on both acid

and alkaline sides. More than 80% of the activity is found in the range pH 4.Oi5.5.

Another unusual feature is the broad optimum activity at the temperature range of

3m50 'C. Xiao Xiao Ping Huang and Colin Monk, 2004 observed carboxymethyl

cellulase with maximal activity at 80°C showing a pH optimum between pH 6.5 and

7.0 and retaining 50% of maximal activity at pH 5.5 and 8.3. Marli Camassola et al.,

2004 identified cellulases from a strain of PeniciNium echinulatum were

characterized for their filter paper activity and B glucosidase activity. Both

activities showed maximum values between pH 4 and 5.

Observations made here are further supported by the findings of Marion et

al., 1991 who reported the multiplicity of endoglucanase in fungus Trichodemta

reesei. Multiplicity of endoglucanase is also reported by Gordon et al., 1985 in

SchizophyNum commune. The literature survey on purification of cellulases

indicated multiple forms of endoglucanases are produced by many microorganisms

(Gum and Brown. 1977; Beldman et al., 1985; Lachke and Deshpande, 1988; Bhat

et al., 1989; SchUlein, 1997). Eriksson and Pettersson, 1975 purified and

characterized five type of endoglucanase in their studies. The existence of multiple

forms of avocado cellulase in crude protein extracts of ripe avocado fruit was

reported by Angelos, 1992. Scrivener and Slaytor, 1994 observed in their studies that

the cellulase of Panesthia cribrata consisted of at least six endo-$-I, 4-D-glucanase

(EC 3.2.1.4) and two P-glucosidase (EC 3.2.1.21) components. Like the mesophilic

Fungi, the thermophilic fungi produced multiple forms of the cellulase components.

Khandke et al., 1989 and Tong et al., 1980, Ramesh Maheshwari, 2000 described in

their studies the two different strains of T aurantiacus producing one form each of

endoglucanase, exoglucanase, and B -glucosidase, but the forms from the two

strains having different properties. The multiplicity of individual cellulases might be

a result of posttranslational and/or postsecretion modifications of a gene product or

might be due to multiple genes. For example, T emersonii produced multiple

endoglucanases, exoglucanases, and $-glucosidases as described by Coughlan et al.,

1988; Coughlan and Moloney, 1988; McHale and Coughlan, 1980; McHale and

Coughlan, 1981; McHale and Coughlan, 1982. Its culture filtrate protein was

resolved by ion-exchange chromatography into four endoglucsnases which, unlike

their variable carbohydrate contents (28 to 5 1°h), had similar molecular masses (68

kDa by gel filtration and 35 kDa by SDS-PAGE), isoelectric points, pH and

temperature optima, thermal stabilities, and specific activities as described by

Moloney, 1985.

Since plant enzymes were generally known to be influenced by mono and

divalent cations, the effect of the presence of ~ g ~ + , ~ n ~ * , colt, cu2*, ca2+, zn2+,

~ i ~ ' , ~i '*, ~ e ~ + a n d ~ g ~ ' at given concentrations are evaluated employing the

endoglucanase enzyme assay. Increase or decrease in the endoglucanase enzyme

activity of each thermophilic isoform due to the presence of a given concentration of

each metal ion or a combination thereof at a given temperature of assay indicated the

influence of the particular metal ion, on the enzyme form. It was found that specific

metal ions influenced the endoglucanase activity at different temperatures and that

the order of influence was Mnzt >co2'>cu2'> Mg2+>Ca2+> zn2+ > ~ i " > ~e''>~i'' >

~ g ~ + .

Similar stimulating effect of Mn2' on endoglucanase from Sclerotium rolfsii

was reported by Lachke, 1982. Hoshino and Ito (1 997) demonstrated that ca2', M ~ ~ ' ,

CO", Mn2+ were essential for themostability of alkaline cellulases from Bacillus sp.

Xiao Ping Huang and Colin Monk, 2004 showed that the reducing agents P

mercaptoethanol and dithiothreitol (DTT) had no stimulatory effect on CMCase

activity and was strongly inhibited by zn2*, H~~~ and the thiol-specific inhibitors.

The substrate specificity studies were suggestive of the fact that both isofoms

exhibited remarkable activity towards carboxymethyl cellulose followed by

cellobiose, chitin and pectin in that order. The enzyme is capable of hydrolyzing

polysaccharides with only B -1,4 linkages, such as carboxymethyl cellulose

confirming the fact that the isolated enzyme is of carboxymethyl cellulase

endoglucanase type.

Endoglucanase enzyme protein stability studies carried out employing

different detergents and the nature of catalytic activity following detergent

interference indicated the degree of stability that these enzyme isofonns could

exhibit from a commercial use stand point.

Needles to say the pepsin and trypsin action on any of these isoform led to

complete loss of endoglucanase activity suggestive of the largely proteinaceous

nature of these enzyme isofonns.

The purification strategy reported here for the isolation and purification of

endoglucanase from the two xerophyre species employed conventional approaches

such as ammonium sulphate precipitation of protein, ion exchange chromatography,

and gel permeation chromatography, to recognize and establish basic characteristics

of the enzyme under these conditions. The cost factor involved in strategizing

immuno-affinity approaches as well as the procedural protocols required to process

expensive import supplies offered significant hurdles and precluded working out

improved methodologies for the isolation and purification needs. Hence

compromises had to be made for optimizing possible methods for our investigative

purpose without having to miss out on the novelties of our observations.

Ammonium sulphate precipitation of protein in cmde extracts has been used

to concentrate and purify endoglucanase enzymes from a variety of sources.

However, ammonium sulphate precipitation of endoglucanase activities during the

fractionation resulted only in partial precipitation. On the other hand, a number of

cellulases and other industrially important enzymes like isomerases, xylanases have

been concentrated by ammonium sulphate precipitation from thennophilic fungi,

such as cellulases from Thermoascur a. riacua (Khandke, 1986) and glucosidases

from S. thennophile (Bhat and Maheshwari, 1987), xylanases from Humicola

lanuginosa (Anand et al., 1990) and Melanocarpus (Prabhu. 1989).

The purification of endoglucanase required various steps such as gel

filtration (Christakopoulos et al., 1995a & 1995b; Bhat et al., 1989), ion exchange

(Wood and McCrae, 1972; Beldman et al., 1985) and affinity chromatography

on Concanvalin-A sepharose (Gong et al., 1979) and on different cellulosic

substrates (Shoemaker and Brown, 1978; Schulein, 1997). Other research workers

had demonstrated application of techniques such as hydrophobic interaction

chromatography (Mansfield et al., 1998; Tomaz and Queiroz 2004), preparative gel

electrophoresis (Wilson, 1988) and Isoelectric focusing in the purification of

cellulases (Sadana et al., 1984). Recently few unconventional techniques such as fast

protein liquid chromatography (Medve et al., 1998), and immunoaffinity

chromatography (Koivula et al., 1996) have been utilized for purification of

cellulases. Ken-ichi Suzuki, 2003 isolated a cellulase (endo- B -1,4-D-glucanase (EC

3.2.1.4) from the hepatopancreas of abalone Haliotis discus hannai by successive

chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl

S-200 HR. The molecular mass of the cellulase was estimated to be 66 kDa by

SDSPAGE and the enzyme showed hydrolytic activity towards carboxpethyl

cellulose with optimal temperature at 38 'C and pH 6.3.

Po-Jui Chenl et al., 2004 isolated and characterized a 94 kDa carboxymethyl

cellulase using DEAE Sepharose anion-exchange column and Phenyl-Sepharose

column with the optimum temperature at 35°C and pH at 7.0, wherein purification

fold was 9.08, and the recovery was 26.4%. and the specific activity was 3.822

Ulmg. In the present study, ion-exchange chromatography on Dowex-1 @H 7.0) was

used successfully, to clearly separate endoglucanase isofoms with approximately 25

fold purification. Krishna-Murti and Stone, 1961 attempted fractionation of a crude

cellulase derived from A. niger by using calcium phosphate and Dowex-l columns.

The purification fold was 28 and 27 for T70 and Too isofoms of Opuntia

vulgaris and that for T70 and Too of Cereusprerogonus were 35 and 31.2 respectively.

The yield of protein obtained for T70 and Tgoof forms Opuntia vulgaris and Cereus

pterogonus following the purification strategy employed here matched well with

earlier reports on endoglucanase purification, and was in the range 17-23% Strategic

improvements in the purification process and the yield of material was possible only

if more modem approaches had been attempted. The lower recovery of the purified

protein is probably due to the sensitivity of certain plant enzymes to extremely low

abundance contaminants like heavy metals present in the chemicals employed for

the isolation methods. Most of the purification protocols resulted in obtaining

homogenous endoglucanase preparations, although the yield obtained was low. This

is because multiple steps were needed in order to get rid of contaminating factors of

endoglucanase activity. Since our interest of study was mainly on thennophilic

thermophilic isoforms, characterization of T70 and Tso were only considered.

Molecular weight detenination of these different isoforms employing gel filtration

technique as well as SDS PAGE technique yielded closely related molecular weight

for each of these species

Molecular weight as Molecular weight as in

Endoglucanase isofoms in Gel filtration SDS PAGE

chromatograph)

Opuntia vulgaris T,o endoglucanase ,50,000 k ~ a

Opunria vulgaris Tso endoglucanase 20,000 kDa

Opuntia vulgaris T,Q endoglucanase 74,000 kDa 66,000 kDa

Opunlia vulgaris Tw endoglucanase 45.000 kDa 36.000 !@a

Cereus prerogonw T ~ o endoglucanase 1,10,000 ma

Cereus plerogonus Tro endoglucanase 45,000 k~~

Cereus plerogonw T70 endoglucanase 74,000 m a 66,000 kDa

Cereus prerogonus Tso endoglucanase 25,000 k~~ 23,000 kDa

The TTO isoform of both species were determined to have a molecular weight of

66,000 Da for the reduced polypeptide band while 36,000 Da for Opuntia vulgaris

T90and 23,000 Da for Cereus pterogonus T90 reduced polypeptide bands were noted.

The molecular weight of 4 isofotms of both plant species as determined by gel

permeation chromatography yielded data, suggested that the probability of subunit

association in the formation of the different temperature isoforms. For example, the

combination of a 45 kDa T ~ o subunit with 20 kDa T5o subunits of the same species

may give rise a 66 kDa T70isoform. Similarly two 66 kDa T70 subunits in association

with a 45 kDa Tpo isoform may give rise to 150 kDa T30 isofom The differences in

the subunit molecular weight of the thermophilic isoforms was indicative of the

basic subunit molecular size that made up each isoform, since differences in the

molecular weight as determined by SDS PAGE and gel filtration was indicative of

the fact that the gel filtration technique generally yielded a higher numeric value for

proteins when they exhibited an extended conformation (non globular) as compared

to their globular nature under SDS PAGE conditions.

The K, and V,, values were obtained (Table I I) from the Lineweaver-Burk

double reciprocal plot. The kinetic feature of the purified endoglucanase employing

carboxymethyl cellulose as substrate was determined. The enzyme endoglucanase

displayed a lower Km for carboxymethyl cellulose, indicative of greater affinity for

this substrate. The rate of hydrolysis on different parts of the substrate may vary. In

the present work, the Km value determined for the enzyme hydrolyzing

carboxymethyl cellulose served to note the amount of substrate required to achieve

half the maximal initial reaction velocity. There is a paucity of information available

on Km values of cellulose hydrolysis by cellulases. Values of 0.5 and 1.6 mgiml

have been published in studies on carboxymethyl cellulose hydrolysis by cellulases

of Mplhecium verrucaria (Halliwell, 1961) and T reesei (Reese & Mandells,

1963).

Denaturation kinetics of each of these isoforms had also employed various

divalent cations to determine the denaturation profile of each isoform through the

temperature range studied. Denaturation kinetics generally reflected through

different time points when assayed at a given temperature. Denaturation kinetics

also helped to determine phase differences in the denaturation profile of an enzyme

protein through a range of temperatures. Further, denaturation profiles can be

utilized to determine the rate of denaturation of a given enzyme protein species (the

denaturation rate constant) which numerical value could then be used to calculate

the change in free energy ( A G ~ that accompanied the given denaturation process or

for each phase of the process. Such an approach enabled to compare and contrast the

denaturation process of an enzyme in the presence and absence of stabilizing agent

such as the divalent cations used in the present study. Determination of free energy

change ( A G ~ for a given denaturation process could then be used to ascertain,

t entrophy changes for the process provided the enthalpy (AH) of the process was

made available or were experimentally determinable.

t In the present study enthalphy (AH) of each denaturation process was

determined by generating the Arrhenius plots (In k vs.liT), whose slopes provided

f the activation energy (Ea) values for use in determining AH. Tabulated results (Table

$ 24) indicated that the AH value for the fast phase of denaturation for the cereus

pterogonus TTO isofom increased nearly two to three fold when 10 mM

concentration of the various metal ions were employed in the temperature range 60 -

s 100°C. while the raise in AH value was only 1.5 fold when a combination of 10 mM

ca2+and 5 mM Mn2+ were used. This trend however was found to be different in the

case of the intermediate phase of denaturation of this same isoform. Estimated

f values for AH indicated a reduction in the numerical value of by a factor of

1.5-1.7 fold, compared to the control intermediate phase AH' values for the

temperature range 60 - 10O0C, independent of whether Mg2+, ca2+ or Mn2+ was

f present during denaturation. Surprisingly the slow phase, AH values were positively

influenced by ca2+ and Mn2+ or their combinations and even the M~*-, Mn2+

f combinations at 10 mM yielding AH values nearly 10 fold greater than that related

to the control or due to 10 mM Mg2' alone in the denaturation process. Wide

f differences were however noted in the fast phase AH values obtained in the case of

Opuntia vulgaris T70 isofonn as noted in the tables, while using the complement of

cations, at identical concentrations. Increase in the intermediate phase A d values of

Opuntia vulgaris TIO isoform was however noted as in the earlier case. While

L moderate increase in the slow phase AH values were noted for this isoform except in

the case where a combination of 10 mM ca2' and 5mM Md' was used that yielded

greater than two fold increase in the numeric value of slow phase AH:

In a similar way, analysis of data obtained for the T9oisoform of Opunria vulgaris

indicated variation for the calculated values at different temperatures in relation

to fast, intermediate, slow phase denaturation profiles for the enzyme as well as for

the enzyme treated independently with 10 mM ca2*, ~ n ~ + a n d the combination of 10

mM Mg2'/5 mM Mn2+and 10 mM ca2*/5 mM Mn2*. What was significant here was

a substantial increase in the energy content of the fast phase due to the presence of

10 mM Mg2'as well as due to the combination of 10 mM ca2+/5 mM Mn2' Slow

phase and intermediate phase stabilization was observed due to the presence of 10

mM ca2', whereas negative enthalpy was noted for the slow phase denaturation of

Opuntia vulgaris when camed out in the presence of 10 mM Mg2'/5 mM Mn2+. In

the case of Cereus plerogonus T ~ o isoform, the control enzyme fast phase wa was noted to be higher than for the other varieties. An increase of tem over the control

was observed for the fast phase when the isoform was denatured through the

temperature 60 - 100°C in the presence of 10 mM ca2'. The significant increase in

1 AH was also seen for the intermediate phase of denaturation of this enzyme species

in the presence 10 mM Mg2+, 10 mM ca2', 10 mM ~ n " , 10 mM ~ ~ ~ ' 1 5 mM ~ n ~ '

and 10 mM ca2'/ 5 mM ~ n " . The substantial increase in the Advalue for the slow

phase denaturation of this enzyme species was seen only in the presence of 10 mM

ca2'/5 mM Mn2'. As for the determination of entrophy Ad, the calculated A4for the

Opuntia vulgaris T70 isoform showed greater numerical value than for A$ for the

intermediate phase of denaturation in the presence of 10 mM each of Mg2* and Mn2+.

The calculated entropy for Cereus pterogonus T,o isoform was noted to be greater

than that for Opuntia vulgaris T70 isoform for the intermediate phase, Similarly AS'

for the 10 mM Mg2' containing intermediate phase was noted to be greater than that

of Opuntia vulgaris Tm. Increase in AS' for the fast phase observed for this

endoglucanase isoform was due to the combination of 10 mM M$+ and 5 mM h4n2'

taken for the reaction. Overall, it was observed that manganese showed a much

greater stabilizing effect for the enzyme.

Comparative analysis of the protein melting temperature using Differential

scanning calorimetry technique noted sharp changes in the protein melting

temperature at 70°C and 90°C for both the isofoms of both plant species confirming

the temperature profile observations made on this isoforms earlier. Ahren (1985)

reported that enzymes are also subject to chemical modifications which may cause a

loss of activity without necessarily resulting in the unfolding of the protein at high

temperature. These include hydrolysis of peptide bonds, deamidation of labile amino

acid residues, and destruction of disulphide bonds. Michael W.Bauer et al., 1999

recognised eglA gene, encoding a thermostable endoglucanase from the

hyperthermophilic archaeon Pyrococcus furiosus, when cloned and expressed in

Escherichia coli. The enzyme had temperature of 100°C and pH optima at 6.0,

respectively a half-life of 40 h at 95'C, and a denaturing temperature of 112°C

which was determined by differential scanning calorimetry.

Fluorescence analysis employing Hitachi-F-4500 Fluorescent

Spectrophotometer noted changes in the intrinsic tryptophan fluorescence

associated with the purified endoglucanase protein. Tlo and Tw isofonns as a

h c t i o n of increasing temperature. The fluorescence intensity of each

endogluc-e species when analysed decreased correspondingly with every 10°C

rise in temperature range 30-10O0C, suggestive of the fact that conformational

changes did occur to the protein molecule subjected to denaturation at each

temperature, eventually burying the tryptophan residues from an initial optimally

exposed conformational state, within the protein structure or within protein

aggregates that found at higher temperature, a . evidence employing Transmission

Electron Microscopy of glutamine synthetases from thermophilic and mesophilic

Bacillus species (Merkler et a]., 1988). Fitter et al., 2001 reported on a comparative

analysis of the thermophilic and mesophilic amylases. Laderman et al., (1993)

reported that the fluorescence emission (L,) of a-amylase from P furiosus at 20°C

exhibited maximum intensity at a (h,,) of 345 nm. When the fluorescent spectnun

was monitored over a range of temperatures, there was no shift observed in the

emission h ,,, suggestive of the fact that the tryptophan residues occupied a polar

environment independent of temperature. A decrease in fluorescent intensity with

increase in temperature is indicated as due to increased quenching caused by greater

thermal motion (Galley and Edeln~an, 1964) in solution.

Detection for the presence of endogeneous manganese as a possible divalent

cation that probably remain associated with these enzyme species, employing

electron paramagnetic resonance analysis, however met with no success due to lack

of the manganese EPR signal at room temperature.

ICP-AES analysis, of ammonium sulphate used for protein precipitation

indicated the presence of chromium (21.43 ppm) in the chemical. Whether the level

of chromium available to the enzyme during the precipitation process affected the

enzyme activity was not specifically determined. Since, the ICP-AES was a recently

established facility, its analytical sensitivity level of < 0.1 ppm for elemental

analysis could not be employed as a co-detection strategy for the determination of

contaminant ions during the purification processes cmied out in this study.

ICP-AES analysis of the tissue homogenate 10,000 x g supernatant also indicated

the presence of MnZ* (19-22 ppm) and M ~ ~ ' (527-568 ppm). The EPR results did

not however establish the presence of manganese in the isoforms studied, probably

due to the relatively lower level of sensitivity of this detection technique. While it

may be contended that manganese did not co-purify with the endoglucanase

isoforms (based on EPR analysis alone), it cannot be emphasized similarly for

diamagnetic elements that remained undetectable by the EPR method, even though

M ~ * * levels were indicated to be greater (by ICP-AES) than Mn2+ levels in the

hornogenate supernatant. Lack of adequate purified protein samples also precluded

wider studies on elemental analysis of the purified material employing the ICP-AES

method, It was therefore believed that the plant enzyme was sensitive to contaminant

factors and/or cold temperature during the purification process and that therefore

resulted in a lower yield of the enzyme activity.

The generation of polyclonal rabbit antiserum against the Tsa isoform of

Opuntia vulgaris endoglucanase aided to investigate cross reactivity between the

antibody and the Cereus plerogonus and the Opuntia vulgaris endoglucanase

isoforms, and indicated regions of commonality in the polypeptide sequences of

these different isoforms. This was confirmed by the observation that the rabbit

antiserum brought about inhibition of the endoglucanase activities during in vitro

test tube assays. Ouchterlony double immunodiffusion, dot-blot and western blot

analysis of endoglucanase isoforms showed cross reactivity between these isoform

activities while they were found differing in their optimum temperature. Western

blot analysis yielded positive bands with polyclonal rabbit anti-endoglucanase

antibody used corresponding to the band positions of T70 and Tgo isoforms of both

species. However, only the CP Tgo exhibited positive band on the blots when

polyclonal rabbit tomato anti-endoglucanase antiserum was employed. These

observations were suggestive of the fact that the endoglucanase antibodies generated

in rabbit recoyised both isoforms possibly due to the common epitopes displayed

for antibody binding by these different isoforms.

Bioinformatics tools aided to identify the conserved sequence regions in the

5' and the 3' -end of selected eukaryotic endoglucanase gene sequences. The

multiple sequence alignment of engA sequences from twenty mesophilic plant

species cited earlier using (Clustal W 1.83) (Fig. 87).

Specific bioinfonnatic tools as cited under 'methods' were used for the

purpose and the existence of conserved domains were recognized in these species.

Oligo nucleotide primer sequences were therefore designed to serve as the 5' end

sense and the 3' end antisense oligos. These primer nucleotide sequences were then

compared with the primer sequences generated by other investigators (Melissa

Goellner et d., 2001) in the data bank that had been used for the amplification of

endoglucanase gene isolated from their various reported sources. Such a comparison

offered a larger opportunity to confirm whether the BMB primers that were

synthesized for the work reponed here, would actually recognize and anneal to the

conserved nucleotide sequences identified for the work in the eukaryotic and

prokaryotic species, to yield the desired PCR amplified product size. The reported

primer nucleotide sequences however enable the generation of PCR products of the

endoglucanase gene despite. significant differences in the localization as well as in

the nucleotide sequences in the conserved regions of the different species considered.

A multiple sequence alignment approach was therefore used to determine the

regions1 segments of complimentarity between the primer sequences and the gene

sequence of endoglucanase from different sources. Inter primer fidelity, G+C

content, primer melting temperature were all determined and compared between the

synthesized BMB primers and the reported counter parts. No regions of

complimentarity were observed between the various BMB primer sequences or

between the primer and the endoglucanase gene sequence excepting in the conserved

sequence domains of the endoglucanase gene for the primers. Yet a PCR product of

1000 bp along with multiple other size hands were obtained making this

investigation deeply difficult, for interpretation, whereas the minimum size of this

eukaryotic gene can be expected to be in the range 1 .O- 1.2 kb (based on a protein

MW 66 kDa). Investigations employing additional experimental approaches are

therefore continuing to establish the fact.

Conclusion

Based on the results obtained in the present study, it is concluded that

b The endo-1, 4-P-D-glucanase enzyme in Opuntia vulgaris and Cereus

prerogonus existed as four isoforms respectively, two being mesophilic,

while the other two being thermophilic isoforms.

b The existence of multiple forms of endoglucanase may be the result of

alternate splicing or due to proteolytic processing of a precursor protein.

2. The T70 and T90 isoforms are independently active for different time periods,

in the temperature range 30 - 100°C, and upto a maximum of 10 min at

100°C in presence of specific metal ions as stabilizing factor.

2. The addition of ~ n ~ ' , ca2- and stabilized the half-life of each of the

thermophilic isoforms in the higher temperatures.

2. The molecular weights of these isoforms were determined to be 66 kDa for

T,,, isoform of Opuntia vulgaris and Cereus pterogonus, 36 kDa and 23 kDa

for TPo isofom of Opunlia vulgaris and Cereus pterogonus..

b Km value for endoglucanase is low for this enzyme activity.

b Temperature dependent denaturation profiles yielded two or three

independent phases for the denaturation process of the thermophilic

isoforms.

9 Differences in the rate constants and thermodynamic properties (Ea, AG AH,

AS) were noted for each isoform undergoing the denaturation process in the

temperature range 30 - 100°C.

9 Immunological cross reactivity and sequence interrelationship existed

between the isoforms.

9 Fluorescence intensity changes detected during the denaturation process

confirmed conformational alterations occurring to the enzyme species. The

intrinsic tryptophan fluorescence decreased with increasing temperature

suggestive of possible molecular aggregation leading to burial of tryptophan

residues within the protein structure.

> Differential Scanning Calorimetry studies yielded information on the melting

temperature (Tm) for the T7o and 7'90 endoglucanase species.

9 Molecular Biology experiments yielded two PCR amplified nucleotide bands

for each of the xerophytic species under study presumably representing the

endoglucanase gene. Work camed out thus far has yielded inconclusive

results to provide information on the nucleotide sequence data for the

thermophilic xerophytic endoglucanase genes.

9 Living systems are considered open systems that can exchange matter and

energy with the environment. Plants remain especially sensitive to the

environmental changes such as water availability, soil and air composition

and light intensity. Changes in the behavior of catalytic activities observed in

our studies due to variations in the divalent cations and their concentration,

and the temperature changes are therefore indicative of the adaptability of

these plant enzymes to different habitats for plant growth. Consequently, the

thermodynamic parameters influenced the adaptability processes within the

plant cell. The metal ion effects observed is suggestive of a regulatory role

for these cations within plant cells, for stabilizing enzyme activities when

influenced by a biotic stress in their habitats.

b Temperature stable plant enzymes should find significant application in

industrial processes.

P Xerophytic plant species are potentially an alternate source of thermostable

enzymes.

b The endoglucanase gene can be employed as a potential marker for the

selection of transgenic plants.