3. RESULTS

The xylose isomerase enzyme activity in the 20% (wlv) cladode homogenate

10.000 x g supernatant o f Cereus pterogonus and Opuntia vulgaris was found to be

2.98 Ulml and 2.55 Ulml respectively. The protein content o f each o f the homogenate

supernatant was determined to be 0.32 mgiml and 0.448 mglml. The specific activity o f

each o f the enzyme preparations was therefore 7.99 Ulmg and 6.78 Uimg respectively

(Tables I and 2).

Temperature profile

Temperature studies employing xylose isomerase activity from each homogenate

source indicated difference in activity at each temperature investigated, as reflected in the

bar graphs (Fig. I and 2). Optimum activity was identifiable from these graphs at two

different temperatures namely. at 60°C (Tm) and at 80°C (T80) for the Cereus prerogonus

enzyme, and at three different temperatures such at 40°C (T40). 70°C (T70) and at 90°C

(Tw) for the Op~mtia vulguri.~ xqlose isomerase activity. In contrast the xqlose isomerase

activity in the leaves o f the mesophilic lxoru species used as a control indicated only a

single temperature optimum for this enzyme activity at 50°C (Fig. 3).

p H profile

pH dependent activity estimates o f xylose isomerase from Cereus plerogonus and

Opuntia vulgaris yielded pH optima at pH 7.0 and pH 7.5 respectively. The enzymes were

found to retain their activity more than 50% in acidic as well as in basic pH ranges (Fig. 4

and 5 ) .

Fig. 1 Effect of temperature on the xylose isomerase activity in Cereus pterogonus. Values are the mean of three independent assays.

Fig. 2 Effect of temperature on the xylose isomerase activity in Opunhh vulgaris. Values are the mean of three independent assays.

Fig. 3 Effect of temperature on the xylose isomerase activity in lxora species. Values are the mean of three independent assays.

M 40 50 W 70 89 90 I00

Temperature CC)

Fig. 4 Effect of pH on the xylose isomerase activity in Cereus pterogonus. Values are the mean of three independent assays.

Fig. 5 Effect of pH on the xylose isomerase activity in Opuntia vulgaris. Values are the mean of three independent assays.

Metal ion study

Studies on the effect of divalent metal ions Mn2', co2', M~~~ and ca2' on the

xylose isomerase enzyme activity of Cereus pterogonus (CPXI) and Opuntia vulgaris

(OVXI) present in their 20% (wlv) cladode homogenate 10,000 x g supernatant when

carried out at selected temperature points in the temperature range 30°C - 100'C, noted the

enzyme to be active at all temperatures studied, for the control as well as for the enzyme

samples containing varying metal ion concentrations (ImM, 5mM and 10mM). Specific

increase in the enzyme activity due to the addition of each metal ion to the reaction volume

was however noted at 40°C, 60°C, 70°C and 90°C, dependent upon the enzyme source. The

optimum concentration of metal ion for use was found to be ImM for ~ g ~ ' & CO", and

5mM for Mn2'. Mn2+ augmented the OVXl and CPXI activity at 70°C as well as at 90°C

and stabilized the enzyme activity at 80°C. The metal ion treated samples of OVXI and

CPXI yielded reduced activity at 100°C. The presence of Mg2' generally reduced the XI

activity at 70°C and 80°C, whereas a moderate increase in the enzyme activity was

observed at 90°C for the sample containing SmM Mg2'. ca2' reduced the XI activity at

90°C. The metal ion activation effect was found to be in the order Mn2'> co2'> Mg2'>ca2'

(Fig. 6 - 13).

Electrophoretic analysis

Electrophoretic analysis of the homogenate 10,000 x g supernatant from Cereus

pterogonus and Opuntia vulgaris were canied out using 7.5% polyacrylamide gels in SDS-

PAGE technique. Both plant species were found to produce similar protein profiles

(Plate 1). Samples were also analyzed by native PAGE and gels were taken for xylose

isomerase activity staining employing 2, 3, 5 triphenyl tetrazolium. Both enzyme species

produced similar type of activity bands having identical electophoretic mobilities (Plate 1).

Fig. 6 Effect of MnCI2 on Cereus pterogonus xylose isomerase activity at different temperatures. Values are the mean of three independent assays.

CPXl CPXl+lmM MnCI, CPX1+5mM MnCI, CPXl+lOmM MnCI,

Fig. 7 Effect of CoC12 on Cereus pterogonus xylose isomerase activity at different temperatures. Values are the mean of three independent assays.

5 CPXl CPXl+lmM CoCI, CPXI+SmM CoCI,

4 - CPXl+lOmM CoCI,

Q 2 3

i 0

Fig. 8 Effect of MgClt on Cereus pterogonus xylose isomerase activity at dimerent temperatures. Values are the mean of three independent assays.

CPXl CPXl+l mM MgCI, CPXI+SmM MgCI, - CPXl+l OmM MgCI, i 6

f i P t P 2

0 30 40 M 60 70 80 90 100

Temperature CC)

Fig. 9 Effect of CaC12 on Cereus pterogonus xylose isomerase activity at different temperatures. Values are the mean of three independent assays.

I CPXl CPXl+lmM CaCI, I CPX1+5mM CaCI,

- 6 CPXl+lOmM CaCI,

{

f 4 j

0

Tomp.r.ture rC)

Fig. 10 Effect of MnC12 on Opuntia vulgaris xylose isomerase activity at different temperatures. Values are the mean of three independent assays.

7 OPXl

6 OPXl+lmM MnCI, OPX1+5mM MnCI,

-- 5 OPXl+lOmM MnCI, f 1 4 3 i3 E 2 E

1

0 M 40 50 MI 70 BO 90 100

Temperatun CC)

Fig. 11 Effect of CoCI2 on Opuntia vulgaris xylose isomerase activity at different temperatures. Values are the mean of three independent ashays.

Fig. 12 Effect of MgClz on Opuntia vulgaris xylose isomerase activity at different temperatures. Values are the mean of three independent assays.

Fig. 13 Effect of CaCI2 on Opuntiu vulgaris xylose isomerase activity at different temperatures. Values are the mean of three independent assays.

6 OPXl OPXl+l mM CaCI, OPX1+5mM CaCI,

- OPXl+lOrnM CaCI,

9 ' i 1'

0

PLATE 1

Electrophoretic analysis of Cereus pterogonus and Opuntia vulgaris homogenate supernatant.

A - SDS-PAGE (7.5% gel) of Cereus pterogorlur and Opuntiu ~~ulguris cladode homogenate 10,000 x g supernatant.

B - Native PAGE (7.5% gel) of the XI enzyme activity in Cereus pteropnus and Opuntiu ~ulguris cladode homogenate 10,000 x g supernatant stained for XI activity.

Lane 1 and 3 - Protein and enzyme activity profile of Cereus pterogonus homogenate 10,000 x g supernatant.

Lane 2 and 4 - Protein and enzyme activity profile of Opuntiu vulgaris homogenate 10,000 x g supernatant.

IF,lectrophoresis was carried out to determine the protein profile of the homogenate supernatant and establish the presence of XI enzyme activity. Molecular weight of the enzyme protein is not

Wgested hem]

Purification of xylose isomerase from CPXI and OVXl

Ammonium sulfate precipitation

Xylose isomerase enzyme activity present in the 10,000 x g supernatant was

initially precipitated with 80% saturation of solid ammonium sulfate at room temperature.

Ion Exchange column chromatography

Subsequently, fractionation of the enzyme activity present in the ammonium

sulphate precipitated and reconstituted sample was camed out, employing Dowex -1

(CI- form) anlon ion exchange column chromatography (bed volume 7011-11) conducted at a

flow rate of 2.5 ml min-'. The column had been previously equilibrated with 50mM

Tris-HC1 buffer (pH 7.0). Following enzyme sample application the column was washed

with approximately S00ml of the buffer until the column effluent was virtually free of

protein (OD < 0.05) as determined by absorbance at 280nm. The column wash was assayed

for the isomerase activity and was found free of this enzyme activity. The adsorbed

proteins bound to the gel were eluted with a linear gradient of 0 - 1.0 M NaCl in a total

volume of 200 ml of the wash buffer. Fractions (3.0 ml) were collected at room

temperature and assayed for protein and xylose isomerase activity in the Cereus

prerogonus and Opuntia vulgaris enzyme fractions (Fig. 14 and 17). Essentially two peaks

(Tho and Tso), of Cereus pterogonus XI eluted at 0.28 M and 0.35 M NaCl concentration,

and three peaks (T40, Tlo and T9,3) of Opuntia vulgaris XI activity eluted at 0.32 M, 0.42 M

and 0.6 M NaCl concentration. The specific activity of the Tm and Tso isoform were 135.4

and 109.9 U mg-' protein, and the specific activity of the T40, T70 and Tw isoform were

106.8, 88.67 and 87.19 U mg-' protein respectively. Enzyme active fractions were pooled

separately, lyophilized and subjected to gel permeation chromatography.

Gel permeation column chromatography

The lyophilized protein was dissolved in 5GmM of Tris-HCI buffer (pH 7.0) and

applied to a column of Sephadex G-100 (20 X 1.2 cm). The column had an exclusion limit

of 90 kDa and was pre-equilibrated with the same buffer. One ml fractions were collected

at a flow rate of 0.5 mllmin and analyzed for protein (A280 nm) and xylose isomerase

activity (Fig. 15, 16, 18 and 19). The specific activity of each enzyme obtained from this

column operation is as illustrated in Tables 1 & 2. The active fractions were pooled for

further studies.

Determination of purity

Fractionation of the xylose isomerase enzyme activity from each source, using

ammonium sulphate precipitation, ion exchange chromatography and gel permeation

technique, yielded enzyme activity, protein content and specific activity as given in

(Tables 1 and 2). CPXI TgO isoform was purified 25.5 fold with a yield of 17.9 %, and the

ThO isoform was purified 20.5 fold with a yield of 16.4%. OVXI Tw isoform was purified

39.2 fold with 20% recovery, and the T70 isofoms was purified 19.6 fold with 14%

recovery. Purity was further determined by electrophoretic analysis of these isoforms on

10% SDS- Polyacrylamide gels (Plates 2 & 3).

The protein samples were analyzed by Natlve-PAGE and visualized by protein and

activity staining for isoforms of CPXl and OVXI. As seen in Plate 4, Native-PAGE

(Lane A to C) gave a single band similar to that obtained in SDS-PAGE (Plates 2 & 3) and

activity staining also showed single protein band (Lane D to F). Consolidation of data

obtained for the purification of the xylose isomerase enzyme activity from Cereus

pterogonus and Opuntia vulgaris suggested that the yield of enzyme from either source

was in the range 32-34% with a combined purification of 46 fold for the isofoms of

Cereus pterogonus. A combined purification of 58 fold was achieved however for the

Fig. 14 Dowex-1 ion exchange column chromatography of 80% ammonium sulphate precipitates resuspension of Cereus pterogonus xylose isomerase activity containing protein sample.

Fraction Number

- Fmtein Prmlb (280 nm) - CPXl iwfm T, (540 nm) - CPXl iwfm Tm (540 nm)

Bed volume - 70 ml Fraction volume - 3 ml No. of fractions - 100 Flow rate - 2.5 d m i n Elution buffer - Gradient elution using 0.1M Tris HCI buffer containing

1 M NaCl

Fig. 15 Sephadex G-100 gel permeation column chromatography of Cereus pterogonus xylose isomerase T60 isoform.

Bed volume -20 ml Fraction volume -2 ml No. of fractions -30 Flow Rate -0.5 ml/ min

0 5 10 15 10 25 30 35

Fnction Number

Fig. 16 Sephadex G-100 gel permeation column chromatography of Cereus pterogonus xylose isomerase Tso isoform.

-a- pmteln profile - - TmaOrefracbm 1 Bed volume Fraction volume No. of fractions Flow Rate

-20 ml -2 ml -30 -0.5 mll min

Fr8ctlon Number

Fig. 17 Dowex- 1 ion exchange column chromatography of 80% ammonium sulphate precipitates resuspension of Opuntia vulgaris xylose isomerase enzyme activity containing protein sample.

Fraction Number

- Protein Profile (280 nm) - OVXI isomrm T, (540 run) - OVXl isofonn T, (540 nm) - OVXl isotorm T, (540 Mn)

Bed volume - 70 ml Fraction volume - 3 ml No. of fractions - 100 Flow rate - 2.5 d m i n Elution buffer - Gradient elution using 0.1M Tris HCI buffer containing

1 M NaCl

Fig. 18 Sephadex G-100 gel permeation column chromatography of Opuntia vulgaris xylose isomerase T7o isoform.

Bed volume Fraction volume No. of fractions Flow Rate

-20 ml -2 ml -30 -0.5 ml/ min

Faction Number

Fig. 19 Sephadex G-100 gel permeation column chromatography of Opuntia vulgaris xylose isomerase TW isoform.

d p r o t s r profile To, s d r e fracbon

Bed volume -20 ml Fract~on volume -2 ml No of fractions -30 Row Rate -0.5 mll min

0.m I ' I '

0 5 1b 15 20 B 30 33

Table 1 Purification of xylose isomerase from Cereuspterogonous.

Step Total Total Specific Purification Yield Protein activity activity fold (%)

(mg) (Units) (Uimg)

1. Cladode homogenate 64.0 5 1 1.55 7.99 I .O 100 2. Ammonium sulfate ppt 26.5 366.34 13.82 1.72 71.6

3. Ion-exchange Chromatography T60 0.9 121.89 135.43 16.94 23.82 T8O 1.2 131.88 109.9 13.75 25.78

4. Gel filtration Chromatography T60 0.51 83.92 164.56 20.58 16.4 T80 0.45 91.92 204.26 25.55 17.96

Table 2 Purification of xylose isomerase from Opuntia vulgaris.

Step Total Protein

(mg)

I. Cladode homogenate 88 2. Ammonium sulfate ppt 48

3. Ion-exchange chromatography T70 1.2 T90 1.65

4. Gel filtration Chromatography T70 0.63 T90 0.45

Total activity (Units)

Specific Purification yield activity fold ("10)

(U/mei

xylose isomerase isoforms of the Opuntia vulgaris (Tables 1 and 2). The purified enzyme

from Opuntia vulgaris and Cereuspzerogonus was found capable of generating

>53% fructose from glucose during 10 min assay at pH 7.5, and at 90°C exhibited. When

xylose was a substrate, the XI enzyme from Opuntia vulgaris was capable of generating

greater than 50% xylulose in a 10 min assay at pH 7.5, while Cereus pterogonus showed

64% ability for the conversion process both at 9OoC.

Molecular mass determination

Both CPXl and OVXI yielded a single polypeptide band on SDS-PAGE analysis.

A linear relationship was initially established by plotting the molecular weight of marker

proteins against their relative mobility on SDS PAGE using the plot. The molecular mass

of Tho and TRO xylose isomerase isoforms of Cereus pterogonus were determined to be

68 kDa, (Plate 2), and that of the T70 and Tm XI isoforms of Opuntia vulgaris were

determined to be 71 kDa and 68 kDa respectively (Plate 3).

Molecular mass of purified xylose isomerase was also determined by gel-filtration

employing Sephadex G-100. A standard plot was established relating molecular mass of

standard proteins with their respective Ve values. The purified xylose isomerase isoforms

were evaluated for their molecular weight against the standard protein using the Ve 1 Vo

ratio of XI isofonns. The apparent molecular mass of T ~ o and Tso xylose isomerase

isoforms of Cereus pterogonus were found to be 66 kDa, and that of the Tin and Tm

isofonns of Opuntia vulgaris were found to be 100 kDa, respectively (Fig. 20 & 21).

Effect of Temperature and pH

The activity of purified CPXI and OVXI isofoms indicated differences at each

temperature investigated as reflected in the line graphs (Fig. 22 & 23). The Cereus

pterogonus isoforms T60 and Tso activity increased considerably with the temperature and

reached its' optimum level at 60°C and 80°C respectively. At higher temperatures, the

PLATE 2

SDS-PAGE (10% gel) of puM~ed xylose isomerase from Ceraus pterogonus.

(8) MW - Protein Molecular weight markers (from Bangalore Genei, India).

Lane A - Purified Tm isoform of Cereuspterogow xylose isomem.

Lane B - Purified TW isoform of Cereusprerogonus xylose isomerase.

(b) Densitometric plot of band width Vs Rf of each protein band detected in the gel. Increase in baseline intensity with increasing Rf value was due to background coomassie stain retain in the gel despite extreme destruning.

PLATE 3

sDS-PAGE (10% gel) of purified xylose isomerase from Opuntia vulgaris.

Profile height MW A B Y

(a) MW - Protein Molecular weight markers (from Bangalore Genei, India).

Lane A - Purified T,o isoform of Opuntici vulgaris xylose isomerase.

Lane B - Purified Tso isofom of Opuntia vulgaris xylose isomerase.

(b) Densitometric plot of band width Vs Rf of each protein band detected in the gel. Increase in baseline intensity with increasing Rf value was due to background coomassie stain retain in the gel despite extreme destaining.

PLATE 4

Native and activity Polyacrylamide gel (10%) electrophoresis of purified xylose isomerase from Cereus pterogonus and Opuntia vulgaris.

A B C D E F

Lane A & D - Purified OVXI T70 isoform

Lane B & E - Purified OVXI TPO isoform

Lane C & F - Purified CPXl T ~ o isoform

Fig. 20 Plot of log mol wt vs VeNo of Cereus pterogonus xylose isomerase isoforms following gel permeation chromatography.

Markers (I) Alcohol dehydrogenase ( I 50,000 Da), (11) bovine albumin (66,000 Da), (111) ovalbumin (45,000 Da), and (IV) carbonic anhydrase (29,000 Da)

Fig. 21 Plot of log mol H.Z vs VeNo of Opuntia vulgaris xylose isomerase isoforms following gel permeation chromatography.

T, 6 T, isoforms of O I X

Markers (1) Alcohol dchydmgenase (150,000 Da), (11) bovine albumin (66,000 Da), (In) ovalbumin (45,000 Da), and (N) carbonic anhydrasc (29,000 Da)

Fig. 22 Effect of temperature on the activity of purified Cereus pterogonus xylose isomerase isoforms Measurements were camed out in triplicate (standard deviations 4%).

Fig, 23 Effect of temperature on the activity of purified opuntia vulgaris xylose isomerase isoforms Measurements were canied out in triplicate (standard deviations 4%).

loo - 90-

0-

g 70-

E " : $j 50-

5 0: 3 . ' 50:

20-

10 -

/m . ,,. . . 9 .

+ T, ~ S O ~ O N I

- P T, lsof0llll \ .

Fig. 24 Effect of pH activity and stability on Cereus pterogonus Tso xylose isomerase isoforms. Measurements were camed out in triplicate (standard deviations 4%).

+control CPXl Metal Ion Stab~llzed CPXl

Fig. 25 Effect of pH activity and stability on Opuntia vulgaris Tw xylose isomerase isoforms. Measurements were carried out in triplicate (standard deviations -3%).

enzyme activity declined rapidly, yielding only 10% of themaximumactivity at 100°C for

the T ~ o isoform and 30% of the optimum for Tso isoform. In case of Opuntia vulgaris

isofoms. T7o and Tw activity increased constantly with the temperature and reached its

maximum level at 70°C and 90°C respectively. At higher temperatures, the enzyme

activity declined rapidly, yielding only 10% of the optimum activity at 100°C for the T70

isoform and more than 50% for Tq0 isofom.

The effect of pH on the T ~ o and Tw isofoms of xylose isomerase was measured in

the pH range 4 - 10 (Fig. 24 & 25). Even though the pH optima of CPXl and OVXl were

found to be 7.0 and & 7.5 respectively while present in the crude homogenate, these

isoforms were found stable in the presence of metal ions and exhibited a broader range of

pH stability between 5.5 and 8.5, but readily denatured at pH values below 4.

Effect of Metal ion

Investigations carried out to determine a role for metal-protein interactions in the

stabilization of thermophilic isoforms employed only the CPXI TRo and OVXI Tw. The

effect of divalent cations used at selective concentrations (ImM, 5mM and IOmM) in the

assay cocktail followed by incubation of the metal-enzyme reaction mixture at 80°C and, at

90°C (corresponding to the temperature optima of these isoforms), yielded xylose

isomerase activity as determined (Tables 3 and 4). For CPXI (T80 isoform), maximum

activity was observed in lOmM Mn2+. Activity in the presence of 5 mM co2' was

approximately 83% and 61% of the activity observed with lOmM Mg2' ions. Similarly for

OVXl (Tw isoform), maximum activity was observed in lOmM Mn2+. Activity in the

presence of ImM co2' was approximately 79% and 61% of the activity observed with 10

mM Mg2' ions. co2+ and Mn2+ were required for xylose isomerase activity, whereas M ~ ~ *

did not enhance enzyme activity in both isofoms. The presence of ca2+, ~ i ~ ' , ~ e ~ ' , cu2+,

Table 3 Effect of divalent cations on Cereuspterogonus xylose isomerase Tso isoform.

Xylose isomerase activity (%)

Sample I mM 5 mM 10 mM

-

CPXI

CPXI + MnC12

CPXI + CoC12

CPXI + MgCI?

CPXI + FeC12

CPXI + CaC12 45.7 33.8 16.9

CPXI + HgCI2

CPXI + ZnClz

CPXI + CUCI?

CPXI + NiC12

CPXI + CdC12 31.3 13.9 4.9

Values in the table represent an average of 3 experimental obsewations (standard deviations 4%)

Table 4 Effect of divalent cations on Opuntia vulgari.~ xylose isomerase Tw isoform.

Xylose isomerase activity (% ifmaximum)

Metal ions 1 mM 5 mM 10 mM

OVXI

OVXI + MnCl2

OVXI + CoClz

OVXI + MgClz 53.8 61.2 61.6

OVXI + FeClz 47.1 43.9 16.3

OVXI + CaCl2 44.8 30.3 13

OVXl + HgCl2 27.1 14.9 5.1

OVXI + ZnCl2

OVXI + CuCl2

OVXI + NiC12 27.5 15.4 3.7

OVXI + CdCh 21.9 11.6 9.3

Values in the table represent an average of 3 experimental observations (standard deviations 4 % )

zn2', cd2' and H ~ ~ ' contributed significantly to the reduction in the xylose isomerase

activity of both the TKO and T90 isoforms.

Effect of Protein denaturants

Investigations to determine the effect of protein denaturing agents such as Urea,

SDS, Guanidine hydrochloride and P-mercaptoethanol, on the xylose isomerase activity of

Cereus pterogonus and Opuntia vulgaris were camed out employing various

concentrations of these protein denaturing agents as shown in Tables 5 and 6. The presence

of 4.OM and 8.OM concentration of Urea on Cereus pterogonus xylose isomerase retained

40% and 32% respectively of its enzyme activity, where as in case of Opuntia vulgaris

xylose isomerase retained 50% activity for 4.OM urea and 27% activity for 8.OM urea were

observed. In presence of SDS on two plant species xylose isomerase (Tau and Tw) clearly

diminished and loss of activity observed when the concentration of detergent increases. In

CPXI, 83% of activity retained when enzyme treated with 20mM SDS whereas 28% of

activity retained in lOOmM SDS. Similarly in OVXI, 78% of activity retained when

enzyme treated with 20mM SDS whereas 15% of activity retained in lOOmM SDS were

observed. But in the treatment of the enzyme with 2M guanidine hydrochloride,

appreciable loss of enzyme activity was observed in both isoforms (Tao and T w ) Finally,

b-mercaptoethanol treatment of both enzyme species exhibited no significant changes in

their enzyme activity.

Determination of Kinetic constants

The kinetic features of the purified xylose isomerase on two different substrates

namely xylose and glucose were determined and the results are compared in Table 7. The

kinetic constant K, and V, values were obtained from double reciprocal Lineweaver-

Burk plots o f enzqme activities at various substrate concentrations. For Tm isofonn of

CPXI, K, and V, were 40mM and 12.9 Ulmg respectively wh ik for Cereus pterogonus

Table 5 Effect of protein denaturants on Cereuspterogonus xylose isomerase Tsa isoform.

Sample Denaturant Xylose isomerase concentration activity (%)

Control CPXI

CPXI+ Urea

CPXI + SDS

CPXI + guanidine hydrochloride

CPXI + P- Mercaptoethanol lOmM

Values in the table represent an average of 3 experimental observations (standard deviations 4%)

Table 6 Effect of protein denaturants on Opuntia vulgaris xylose isomerase Tw isoform.

Sample Denaturant Xylose isomerase concentration activity (%)

Control OVXl 100

OVXl+ Urea

OVXI + SDS

OVXI + guanidine hydrochloride 0.1M 0.5M 2.0M

OVXl + P- Mercaptoethanol lOmM 94.0

Values in the table represent an average of 3 experimental observations (standard deviations 4 % )

Fig. 26 Lineweaver-Burk double reciprocal plot of Cereus pterogonus xylose isomerase isoform activity using xylose as substrate.

Fig. 27 Lineweaver-Burk double reciprocal of Cereus pterogonus xylose isomerase isoform activity using glucose as substrate.

Fig. 28 Lineweaver-Burk double reciprocal of Opuntia vulgaris xylose isomerase isoform activity using xylose as substrate.

Fig. 29 Lineweaver-Burk double reciprocal of Opuntia vulgaris xylose isomerase isoform activity using glucose as substrate.

Table 7 Comparison of Kinetic constants for CPXI and OVXI isoforms.

Particulars Substrate Temp. Km ("C) (mM)

Vmax VmaxIKm (Utmg)

CPXI Xylose 60

isoforms 80

Glucose

OVXI Xylose

isoforrns

Gluwse

glucose isomerase they were 102mM and 7.75 Ulmg. where as for Tso isoforms of CPXl

K, and V,, were 30mM and 8.72 Ulmg respectively while for Cereus pterogonus glucose

isomerase they were 121mM and 6.89 Ulmg. Similarly for T70 isoforms of OVXI, K, and

V,,, were 27.4mM and 9.76 Ulmg respectively while for OV glucose isomerase they were

1 1 1.4mM and 7.02 Ulmg. whereas for Tw isoforms of OVXI, K, and V,, were 46.4mM

and 9.88 Uimg respectively while for OV glucose isomerase they were 259mM and 9.1

Uimg. All four isoforms of xylose isomerases from the two different plant species

displayed a lower K, value for xylose than for glucose, while the apparent V,, value of

these isoenzymes with xylose as substrate were higher than those with glucose

(Fig. 26 -29).

Thermal inactivation of xylose isomerase

Thermal inactivation of the T70, T80 and Tw thermophilic XI isoforms were carried

out using the enzyme protein with a specific activity 133, 204 and 266 Uimg respectively,

and in the temperature range 60°C-100°C. All isoforms were completely stable for 30 mins

at or below 60°C. When the logarithms of the residual activities of T70, T80 and Tw

isoforms at a given temperature were plotted against reaction time, all isoforms showed

linear inactivation kinetlc curves (Fig. 30-41). However, denaturation plots of activity vs

time indicated multiple phases (Fast, Intermediate and Slow) in the denaturation profile of

each isoforms (Fig. 42). CPXl and OVXI enzyme thennostability was first characterized in

the absence of metal ions. Under the conditions, Tso isoform of CPXI had a half-life (tin)

of 42 rnin at 80°C, 29 rnin at 90°C and 17 min at 100°C, where as the T70 isoform of OVXI

had a half-life of 37 rnin at 70°C, 27 min at 80°C, 16 min at 90°C and 10 rnin at

10O0C, and the TW isoform of OVXI had a half-life (tl12) of 22 min at 80°C, 17 rnin at 90°C

and 13 rnin at 100°C. All isofoms of CPXl and OVXI exhibited a first-order type

inactivation profile. The denaturation rate constant obtained for all the three different

Fig. 30 Denaturation kinetics of control Cereus pterogonus xylose isomerase Tsa isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co-efticients r? above 0.96.

Time (min) E - e n ~ y m e acuvlty after heat~ng time I at a given temperature E,, - rnlyme activlty at zero heat~ng time.

Fig. 31 Denaturation kinetics of ~ n ' ' treated Cereus pterogonus xylose isomerase Tw, isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co-efficients r' above 0.96.

Tlma (mln) E - enzyme activity after heating tlme t a t a g ~ v e n temperature E,, -enzyme activity at zero heating time.

Fig. 32 Denaturation kinetics of co2+ treated Cereus pterogonus xylose isomerase Tm isoform at different temperatures as a function of time. Vertical bars indicate standard error ( ~ 3 ) . All linear regression had correlation co-efficients r' above 0.96.

< . , . , . , . , . 8 . , . 4 0 5 10 15 20 25 30 35

Tlms (mln) E - cri/ylne activlty after heaung time t at a given tcmperaturz E,, - cnryme act~vity at zero hcatlng ! m e .

Fig. 33 Denaturation kinetics of M ~ * + treated Cereus pterogonus xylose isomerase T80

isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co-efficients r' above 0.96.

, . , . , . , . I . I . I .

0 5 10 15 20 25 30 3!

Tim (mln)

E - enzyme activity after heating time tat a given temperature E,, - enzyme activity at zero heating time.

Fig. 34 Denaturation kinetics of control Opuntia vulgaris xylose isomerase T70 isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co- efficients i above 0.96.

0.0 0 5 I0 15 20 25 30 35

Time (min)

E - enlymc acrlrlty allcr heat~ng lime t ar a given tempcraturr E, - en7ymc sct~vity al le ro hearing ume.

Fig. 35 Denaturation kinetics of ~ n ~ + treated Opuntia vulgaris xylose isomerase T,o isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co-efficients r' above 0.96.

E -enzyme activity after heat~ng time t at a given temperature E,, - enzyme activity at zero heating time.

Fig. 36 Denaturation kinetics of co2+ treated Opunfia vulgaris xylose isomerase T70 isoform at different temperatures as a function of time. Vertical bars indicate standard error ( ~ 3 ) . All linear regression had correlation co-efficients ? above 0.96.

Fig. 37 Denaturation kinetics of M~' ' treated Opuntia vulgaris xylose isomerase T70 isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co-efficients r? above 0.96.

4 0 ' ~ (t ,= 438) 6O0C(t,=54)

A 70°c (t ,= 65) 1 5 - i 7 BO'C (t,= 36)

Time (mm) E - enzyme activity afier heating time I at a glven vmpralurt: E,, -enzyme activity at zero healing time.

3 . 1 0 -

8 - I .

0 5-

oo-, 0 5 10 15 20 25 30 35

Time (rntn) t e r l ~ y l n ~ ~ c 1 1 \ 1 t y after herllng llmr I at a pl\cn te~llprrdture E rnLymc dctlrllr dl zero hrdllng tlmc

9

f

. . , . , . , . , . , . : .

90°C (t ,= 12)

4 1m0c (t,= 10)

Fig. 38 Denaturation kinetics of control Opuntia vulgaris xylose isomerase Tw isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co- efficients r2 above 0.96.

E - enl jmc acrl\lty after heat~ng ltme t at a y v e n tcrnpcrarurc E,, - ervyrnc a c t ~ r ~ t y at Len1 heat~ng tlrnc.

Fig. 39 Denaturation kinetics of ~ n ' ' treated Opuntia vulgaris xylose isomerase Tw isoform at different temperatures as a function of time. Vertical bars indicate standard error (nr3). All linear regression had correlation co-efficients r' dbove 0.96.

E - enzyme activity after heating time t at a given temperature E, -enzyme activity at zero heatin! time.

Fig. 40 Denaturation kinetics of coU treated Opunfzh vulgaris xylose isomerase Tw isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co-efficients above 0.96.

Time (min) E - enzyme acuvlly atlcr heaunp flme r at a glven temperature E - cn7yme acllvlry at ,era hcatlng llmc.

Fig. 41 Denaturation kinetics of M ~ ' + treated Opuntia vulgaris xylose isomerase Tw isoform at different temperatures as a function of time. Vertical bars indicate standard error (n=3). All linear regression had correlation co-efficient r' above 0.96.

E - enf.yme activity after heating time t at a given temperature El - enzyme actlnty at zero heating lime.

Fig 42 Denaturation kinetics of CPXI and OVXI thermophilic isoforms observed at 100°C.

2 1 , ' ; . , !,I I-.

0

0 5 (0 15 20 25 30 Tb. (dm)

T8o isoform of CPXl

-c mnbM r - Cd' beatec 4- Wfmatea t Um" haled

T70 isoform of OVXI Tw isoform of OVXI

Note: Observed the phase differences (Fast, intermediate, and slow phases) in the denaturation profile of each isoform

xxiii

isozymes are represented in the Tables 8-10. The Arrhenius plot of denaturation rate

constant (k) for 1/T for the Tso isoform of CPXI and the T70 and Tuo isoforms of OVXl

were found to be linear (Fig 43-45). Activation energy (Ea) for the thermal inactivation of

each isoforms (T70, T80 and T90) was determined from the Arrhenius equation and was

found to be 61.84, 58.76 and 66.639 Jlmol respectively.

To determine the metal requirement of each isofonns (T70, TgO and Tw) for

thermostability, the apoenzyme was incubated in the presence of 1 mM co2', 10 mM Mn2'

and 10 mM Mg2' at different temperatures and for various periods of time. Residual

activity was measured with xylose as the substrate in the presence of CoCI2. The

apoenzyme was significantly less stable than the enzyme containing co2+, Mn2+ or M$i.

Of the three metal cations, Mn2' significantly stabilized all three isozymes. coZ+ was less

stabilizing than Mnzi, but co2+ was found to be stabilize the isozymes at higher

temperature (Tables 3 & 4). MgZ' was significantly less efficient than the other two metals

at stabilizing the three isozymes. The Ea values for the three isozymes were calculated both

in the presence and the absence of metal ions (Table 14). The nature of the metal ion

employed clearly affected the Ea of each isofonns. It was remarkably high in the presence

of co2' or Mn2+. Since the Ea for denaturation and the temperature at which the enzyme

starts denaturing at a measurable rate, increased from the apoenzyme to the Mn2+-

containing enzyme, loss of the metal cofactor wuld be considered a limiting step in the

isoforms inactivation process. Higher Ea values for denaturation of the co2'- enzyme and

the M n 2 + a y m e reflected the higher thermal energy that these metalknzyme complexes

need to accumulate before losing the tightly bound metal, causing them to unfold. The

higher Ea for denaturation provided by co2' and Mn2+ compared with that provided by

Mg2' reflect the different binding affinities these cations exhibit for the xylose isomerase

lsozymes.

Fig. 43 Arrhenius Plot of denaturation of Cereus pterogonus Tso isoform (Temperature range 4O0C-100°C). All linear regression had correlation co-efficients above 0.95.

Fig. 44 Arrhenius Plot of denaturation of Opuntia vulgaris Tm isoform (Temperature range 40°C-100°C). All linear regression had correlation co-efficients ? above 0.95.

2 -

3 -

4 - L .

4 5 -

4 -

7 -

¤ Ea =58.-6 x 10' J mol

m

. 2 6 2 7 2 8 2 9 3 0 3 1 3 2

Ea - actlvatlon energy k - react~on rate constant T - absolute temperature

3 3

Ea - activation energy k - reaction rate constant T - absolute temperature

Ea = 61.84 I 10' J b o l

2.6 I:? 2 2 2.) 3.0 3.1 3 1 31

1 m ( 1 o Y 1 )

4 -

-' .5-

I 4-

7 -

h, hx..

\ r \\,,

\\ \\

'\

Fig. 45 Arrhenius Plot of denaturation of Opuntia vulgaris Tw isoform (Temperature range 40°C-100°C). All linear regression had correlation co-efficients ? above 0.95.

Ea - activation energy k -reaction rate constant T - absolute temperature

According to the transition state theory, the rates of inactivation observed in the

temperature range yield the activation enthalpy and entropy. The increased slope and the

higher Y intercept suggest increased activation energy and a larger pre-exponential factor

due to the addition of the bivalent ion. Tables 8-20 list the observed inactivation rate

constant, deduced activation energy, enthalpy, entropy and Gibbs free energy. These

parameters yielded insights into the nature of the inactivation process. The activation

enthalpy AH, is in agreement with previously measured activation energies for the

unfolding of soluble proteins. Activation Gibbs free energies AG, which is measures of the

spontaneity of the inactivation processes, is lower than the AH values. This is due to the

positive entropy generated during the inactivation process. The activation entropy

represents the difference in the extent of local disordering between the transition state and

the ground state for the inactivation pathway. Thus, the positive AS are in agreement with

increasing local disorder in the transition state when compared with the ground state. The

increasing value of AS as a function of metal ion concentration is considered to be due to

the degree of disorder in the structure required to reach the transition state for the unfolding

process since the structural packing in the ground state probably remained firmly stabilized

by the bivalent ion.

The rate of denaturation of each isoforms was then determined from the slope of the

tangent drawn and was estimated as a ratio of their YIX values Consequent to obtaining the

denaturation rate constants for each isoforms at each temperature studied, the free energy

change (AG) related to each denaturation phase was calculated by supplementing the

experimental rate constant values obtained in the relationship AG = - RT Ink (where k =

denaturation phase rate constant) (Tabla 11-13).

The denaturation experiments were followed with determination of enthalpy and

entropy values for each of the denaturation isoforms exhibited by the two thmoph i l i c

Table 8. CPXl TED isoform denaturation rate constants (k). (Determined in presence of divalent cations)

Temperature Enzyme + Enzyme + Enzyme + e c )

Enzyme 1mM CoC12 lOmM MnCll lOmM MgC12 x x 1w2 x lo-* x 1v2

-~

Values are given as Mean i SE (n=3), Rate constam (k) values are exprmsed in mo le .~~ ' . s ec~ ' .

Table 9. OVXI T70 isoform denaturation rate constants (k). (Determined in presence of divslent cations)

Temperature Enzyme + Enzyme + Enzyme + e c )

Enzyme ImM CoC12 1OmM MnCll 10mM MgC12 x 10.' x 10.' x 1v2 x 1w2

40 0.177 * 0.014 0.158 i 0.015 0.224i 0.012 0'239* 0'022

60 1.802*0.196 1.281 i0.088 1.635*0.105 0.936i0.062

100 6.863i0.883 6.814*0.683 6.684k1.15 6.079k0.57

Values are given as Mean f SE (n=3), Rate constant (k) values are expressed in mo~e.l".sec-'.

Table 10. OVXl Tw isoform denaturation rate constants (k). (Determined in presence of divalent cations)

Temperature Enzyme + Enzyme + Enzyme + e c )

Enzyme 1mM CoClt lOmM MnClt lOmM MgC12 x 10.2 x 10-2 x x lo.=

Values are given as Mean * SE (n=3), Rate constant (k) values are expressed in rnole.~' .seL' .

Table 11 Calculated Free energy changes (AG) for CPXI Tso isoform at different temperatures in presence of divalent ions

Control Temperature

Enzyme + Enzyme + Enzyme + e c )

Enzyme 1mM CoC12 lOmM MnCLl 1OmM MgC12

100 2.385i0.883 2.771 i0.683 2.092i 1.15 2.280i0.57

Values arc given as Mean + SE (n=3), AG values are expressed in KJ/mol.

Table 12 Calculated Free energy changes (AG) for T70 isoform of OVXI at different temperatures in presence of divalent ions

Temperature Control Enzyme + Enzyme + Enzyme + e c )

Enzyme ImM CoClz lOmM MnCll lOmM MgC12

Values are glven as Mean i SE (n=3). AG values are expressed in KJImol

Table 13 Calculated Free energy changes (AG) for Tw isoforms of OVXI at different temperatures in presence of divalent ions.

Control Temperature Enzyme + Enzyme + Enzyme + e c )

Enzyme 1mM CoC12 1OmM MnC12 10mM MgCI?

100 2.174i0.883 2.262i0.683 2.103i1.15 1.962iO.57

Values are givcn aa can f SE (n=3), AG values are expressed in KJimol.

Table 14 Calculated activation energy (Ea) for OVXl and CPXI isoforms at different temperatures in presence of divalent cations.

Opuntia vulgaris

Particulars Cereus pterogonus Tao isofoms

T,n isoform Ton isoform

Control Enzyme (~.mol") 61.841 0.014 66.63 1 0.015 58.76 * 0.022

Enzyme + ImM CoClz 59.52 1 0.196 81.2931 0.088 64.9571 0.062

(~.rnol.') Enzyme + lOmM

MnC12 53.81 1 0.155 76.07 * 0.304 68.21* 0.057 (J.rnol.')

Enzyme + I OmM MgC12, 52.0811 0.189 50.5225 0.1 1 46.862* 0.048

(J.mol- )

Values are given as Mean 1 SE (n=3). values are expressed in Jlmol

Table 15 Calculated Enthalpy (AH) for CPXI (T80 isoform) at different temperatures in presence of divalent cations.

Temperature Control Enzyme + Enzyme + Enzyme +

e c ) Enzyme 1mM CoC12 IOmM MnCIZ IOmM MgCI2

Values arc given as Mean * SE (n=3), AH values are expressed in KJlmol.

Table 16 Calculated Enthalpy (AH) for OVXl (T70 isoform) at different temperatures in presence of divalent cations.

Temperature Control Enzyme + Enzyme + Enzyme + e c )

Enzyme 1mM CoCh IOmM MnC12 lOmM MgCll

Values are glven as Mean i SE (n-3). AH values are expressed in KJimol

Table 17 Calculated Enthalpy (AH) for OVXI (T90 isoform) at different temperatures in presence of divalent cations.

Temperature Enzyme + Enzyme + Enzyme + ec) Enzyme 1mM CoCll 10mM MnC12 10mM MgC12

Values ginn as Mean + SE (n=3), AH values are expressed in KJImol.

Table 18 Calculated Entropy (AS) for CPXI (Too isoform) at different temperatures in presence of divalent cations.

Temperature Control Enzyme + Enzyme + Enzyme +

e c ) Enzyme ImM CoClz 1OmM MnC12 1OmM MgC12

100 0.142i0.883 0.158i0.683 0.16811.15 0.111i0.57

Values are given as Mean * SE (n=3), AS values are expressed in ~.I.rnol~'.~~'.

Table 19 Calculated Entropy (AS) for OVXl fl70 isoform) at different temperatures in presence of divalent cations.

Temperature Control Enzyme + Enzyme + Enzyme +

e c ) Enzyme 1mM CoClz 10mM MnC12 10mM MgClZ

100 0.152i0.883 0.14510.683 0 3 l l S 0.12510.57

Values are given as Mean + SE (n=3), AS values are expressed in ~J.mol''.K-'.

Table 20. Calculated Entropy (AS) for OVXI (Tw isoform) at different temperatures in presence of divalent cations.

Temperature Control Enzyme + Enzyme + Enzyme + e c )

Enzyme 1mM CoC12 lOmM MnC12 lOmM MgCll

xylose isomerase plant species. Arrhenius plots were generated for each isoforms

independently, (Fig. 43-45) using the experimental rate constant (K) in the relationship,

In k vs IlT, for initially obtaining the activation energy (Ea) values (Table 14), and then

substituting the Ea value in the equation AH = Ea-RT (Tables 15-1 7). Since both AG and

AH values were determinable, the entropy (AS) of each of the denaturation process was

estimated by supplementing the experimentally determined AG and AH values in the

established thermodynamic relationship AG = AH - TAS (Tables 18 - 20).

Differential Scanning Calorimetry studies

The thermally inducible denaturation process for the purified xylose isomerase

isoforms (Two of CPXl & Tw of OVXI) was canied out in the absence and presence of

metal ions employing the Differential Scanning Calorimetry technique (DSC) (Fig. 46-49).

The melting temperature (T,, the temperature maximum of the heat capacity) for purified

Txil and Twl isoforms in the absence of added metal ions was found to be 80°C and 90°C.

whereas in the presence of divalent metal ions (5mM of ~ n ~ ' and ImM of co2+) the

enzyme exhibited a thermal transition with the T,, increasing to above 100°C for both

isoforms.

Spectrofluorimetric determination

Fluorescence measurements detected changes in the intrinsic fluorescence of the

T8o and TW xylose isomerase isoforms when determined in the temperature range 40°C -

100°C. Tryptophan fluorescence showed substantial decrease with increase in temperature.

The fluorescence emission was monitored using a spectrofluorimetry in the wavelength

range 280-400111x1 employing a single h excitation of 280nm. The fluorescence spectrum

for each of the isofoms is as observed in (Fig. 50 & 51). Shifts in the emission & were

observed for each of the isoforms as a function of the temperature with the fluo~escence

peak high for 30°C and low for 100°C.

Fig. 46 Differential Scanning Calorimetry of Cereus pterogonus xylose isomerase (Tso isoform).

Fig. 47 Differential Scanning Calorimetry of Cereuspterogonus xylose isomerase (Tao isoform) in presence of MnC12 (5mM) and CoC12 (1mM).

Fig. 48 Differential Scanning Calorimetry of Opuntia vulgaris xylose isomerase (Tw isoform).

4 , . , , , , , , , , , , , , , l J O a ~ 6 0 l O M 9 0 1 0 0

Temperature (C)

Fig. 49 Differential Scanning Calorimetry of Opuntia vulgaris xylose isomerase (Tqo isoform) in presence of MnCIz (5mM) and CoC12 (1mM).

J . . . . , . , . , 1 JO 4 m eu m m oo roo 110

Twnprmm CC)

Fig. 50 Fluorescent spectroscopic analysis of Cereuspterogonus Tao xylose isomerase isoform.

Fig. 51 Fluorescent spectroscopic analysis of Opuntia vulgaris Tw xylose isomerase isoform.

EPR analysis

The presence of endogenous Mn2' and coZ' associated with each isoform of CPXl

and OVXI were investigated employing the Electron Paramagnetic Resonance (EPR)

technique. The presence of endogenous Mn2+ and co2' was not detectable by EPR analysis

at room temperature (Fig. 52). Both paramagnetic divalent cations were however detected

in EPR at liquid nitrogen temperature (Fig. 53 & 54). Mn2+ contains six resonance line

(1=512) but the spectrum contained only a single resonance line due to dipolar-dipolar

interaction, whereas in the case of co2', eight expected resonance lines were obtained

(1=712). The g values and Hyperfine value (A) were calculated from the EPR spectrum

obtained for all isofoms of CPXI and OVXI respectively (Table 21).

ICP-AES analysis

Inductively Coupled Plasma - Atomic Emission Spectroscopic (ICP-AES) analysis

of (a) the xerophyte habitat soil. (b) distilled water used for the experimental purpose, (c) a

sample of the commercial ammonium sulphate used for protein precipitation and (d) the

cladode homogcnate 10,000 x g supernatant, for the detection of the presence of specific

elements, in the samples like divalent cations, heavy metals and phosphorus, indicated the

presence of relatively higher amounts of Mg2' (527 ppm - 568 ppm) and Mn2' (19 ppm -

23 ppm) along with P (19 ppm-187600 ppm) in the plant cladode 10,000 x g supernatant.

The presence of chromium as a heavy metal was also detected in the commercial

ammonium sulphate used for protein precipitation (Table 22).

lmmuno reactivity Studies

lrnmunoreactive studies indicated that polyclonal rabbit anti-xylose isomerase

antiserum was capable of inhibiting the xylose isomerase activity greater than 70% when

Tw used in the enzyme assay. However the TbO. T70 and Tw isofoms was found inhibited

only to the extent of 25-55 % using the same antiserum (Fig. 55).

Fig. 52 EPR Spectrum for detection of ~ n " in Cereus pterogonus and Opuntia vulgaris xylose isomerase at room temperature. (a) Distilled water (9.39 GHz), (b) Tris-HC1 buffer (y = 9.09 GHz), (c) ImM MnCll (y = 9.19 GHz), (d) Tau CPXI (y = 9.39 GHz), (e) T90 OVXI (y = 9.35 GHz).

Fig. 53 EPR Spectrum of ~n'' and co2+ in purified Cereus pterogonus xylose isomerase detected under liquid nitrogen temperature. (a) Tris-HCI buffer (7 = 9.067 GHz), (c) ImM CoClz (y = 9.075 GHz), (d) Tao CPXI (y = 9.076 GHz), (e) Tso CPXI (y = 9.075 GHz).

Fig. 54 EPR Spectrum of ~ n " and CO" in purified Opuntia vulguris xylose isomerase detected under liquid nitrogen temperature. (a) Tns-HC1 buffer (y = 9.067 GHz), (c) ImM CoC12 (y = 9.075 GHz), ( d ) T70 OVXI (y = 9.076 GHz), (e) Too OVXI (y = 9.075 GHz).

Table 21 Hypertime (A) and g-value of ~ n " and co2+ detected by X-band EPR analysis in OVXI and CPXl samples.

Particulars g values g values Hyperfine value

(g 1) (g 11) for CO*+(A)

(mT)

co2' control 2.297 135

~ n ~ ' control 2.077

CPXl T a isofom 2.071 2.3046 130

Tso isoform 2.075 2.309 133

OVXI T,o isoform 2.069 2.304 132

Tw isoform 2.079 2.305 133

Table 22 ICP-AES analysis of soil, water, cladode homogenate and ammonium sulphate for the quantitative determination of elements in the samples.

Particulars Distilled Ammonium Cereus Opuntia H z 0 sulphate Pterogonus Vulgaris Soil

Copper ( P P ~ ) 0.003 ND 6.6 5.28 ND

Iron (ppm) ND 11.09 9.96 6.40 11,032.58

Magnesium ( P P ~ ) ND 0.383 527.1 1 568.93 1225.3

Manganese (ppm) ND 2.415 22.55 19.04 252.49

Cadmium (ppm) ND 0.165 0.179 0.174 4.92

Cobalt (ppm) ND 0.205 0.279 0.230 15.45

Zinc (ppm) ND 0.288 4.453 4.81 26.90

Chromium (ppm) ND 21.42 16.52 16.84 1706.54

Nickel (ppm) ND 5.381 6.726 9.83 294.03

Lead ( P P ~ ) 0.0006 ND ND ND 2.3

Phosphorous (%) O.l 28 0.67 18.76 11.52 0.681

Calcium @pm) ND ND 245.9 50.48 322.4

*ND -Not Detectable

Purified xylose isomerase isoform was used to generate polyclonal antibodies in

male rabbit as reported under methods. The rabbit antiserum was used as the antibody

source for experiments involving Ouchterlony agar double immuno diffusion, against

purified CPXI and OVXI isomerase isofonns. As shown in Plate 5, the purified xylose

isomerase isofoms showed a precipitin line indicating that the serum used was specific to

xylose isomerase. A more sensitive dot-blot analysis was also performed. As illustrated in

Plate 6 rabbit antiserum to the Tw isoforms of OVXI reacted with all XI isoforms namely

(Tm, T70, T ~ o and Tq,). This observation was further confirmed by western blot analysis

following SDS-PAGE of these isoforms. A clearly distinct immuno detectable band was

observed in Plate 7 for all forms of xylose isomerase and exhibiting a molecular mass

around 68 kDa. These observations suggested that all XI isoforms did have common

epitopes or exhibited partial structural homology.

Bioinforrnatics Studies

Multiple sequence alignment of q l A sequences from 11 sources (thermophilic

bacteria and mesophilic plants) was carried out using ClustalW (1.83). Amino acid

sequences were aligned to determine homologous regions, and identifying particular amino

acids that may probably be responsible for increased thennostability. Relatively few

regions of homology were however recognized (Fig. 56). The amino acid sequences

involved in substrate binding, metal ion binding as well as in catalytic hnction were found

to be completely conserved. A cis peptide linkage involving adjacent glutamic acid and

proline, responsible for the formation of a rigid structure at the active site was found to be

well conserved in all the XIS studied. The amino acids were not located within a single

domain, but were distributed along the length of the primary structure. The essential

Structure at the catalytic site of XI appeared to be analogous in all the species that were

compared. Apparently, despite of low homology, the sequences seemed to form similar

Fig. 55 Inhibition assay for Cereus pterogonus and Opuntia vulgaris xylose isomerase isoforms using polyclonal anti xylose isomerase rabbit antibody serum

Control Enzyme 0 Control E w e + Ab Treated

PLATE 5

Ouchterlony immunodiffusion studies employing the xylose isomerase isoforms of Cereus pterogonus and Opuntia vulgaris using polyclonal antixylose isomerase rabbit antibody serum.

Label A - Purified CPXI Tm isoforrn (protein 264 pglml)

Label B - Purified CPXI Tgo isoform (protein 225 pg/ml)

Label C - Purified OVXI T70 isoform (protein 250 pg/ml)

Label D - Purified OVXI TW isoform (protein 250 pg/ml)

Label E - Polygclonal rabbit anti XI antisera (protein 300 pg/ml)

PLATE 6

Dot-blot analysis of xylose isomerase enzyme preparation using rabbit polyclonal OVXI antibody serum.

A - Purified Cereus pterogonus xylose isomerase isoforms

Dot I - Distilled water Dot 2 - Txo isoform. Dot 3 - Tw isoform.

B - Purified Opunria \ulgaris xylose isomerase isoforms

Dot I - Distilled water Dot 2 - TW isofonn. Dot 3 - T7o isoform.

I: 250 dilution of xylose isomerase 2.0 @g antibodies were used.

PLATE 7

Western-blot analysis of xylose isomerase using rabbit polyclonal OVXI antibody serum.

MW - Protein Molecular weight markers (from Bangalore Genei, India).

Lane 1 - Purified TN) isoform from Cereus pterogonus

Lane 2 - Purified Tsn isoform from Cereus pterogonus

Lane 3 - Purified T70 isoform from Opuntia vulgaris

Lane 4 - Purified Tyg isoform from Opunria iwlgaris

Lane 5 - Crude extract of Cereus pterogonus

Lane 6 - Crude extract of Opuntia vulgaris

Fig. 56 Homology alignment of the XyL4 sequences of 11 species. The XylA protein sequences, deduced from the nucleotide sequences obtained from the EMBL database were analyzed with the CLUSTAL W program (1.83) for Multiple Sequence Alignment.

-------...--.-......-----...--..-------...- ..--------..---------------...---....-.---.

MNKYFEN 7 MNKYFEN 7

..................................................

..................................................

........................... ------.--.-.--.-. -----....-----..---------------..-.---...~~

MEYFKN 6

........................................... MREYFEN 7

--------..--------.-----..-...--....----... MAEFFPE 7 I lhDFPDS ?

T.saccharo1ytrcum. VSKIKYEGPKSNNPYSFKFYNPEEVIDGKTMEEHLRFSIAYWHT~ADGT 57 Thermoanaerobacter~m VSKIKYEGPKSNNPYSFKFYNPEEVIDAKTMEEHLRFSIAYWHTFTADGT 57 T.thermosulfurogenes -----.------....------..---------.--------..-----.

T,ethanollcus -~-----~-~~~~.-~~.~~.....------~~.-~-~-~.....~~~-- T.yonseil. VPKVQYEGPKSNNPYAFKFYKPDEIIDGKF'LKEHLRFAVAYWHTFTTT 56 C.bei?erlnckrl VSKINYEGANSKNPYSFKYYNPDEIIGDKAMKEHLRFALSYWHTLTATGA 57

IPKIOFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFJNEGR 57

T . ~ a c c h a r o l y t ~ c ~ m . DQFGKATMQRPWNHYTDPMDIAKRRVEAAFEFFDKINAPFFCFHDRDIAP 107 Therrnoanaerobacteriw DQFGWTMQRPWNHYTDPMDIAKARVEAAFEFFDKINAPFFCFHDRDIAP 107 T.therrnosulfurogenes ---GKATMQRPWNHYTDPMDIAKARVEARFEFFDKINAPYFCFHDRDI~P 47 T.ethanolicus .--. APTMORPWDHFTDPMDIAI(ARVEAAFELFEKLDVPFFCFHDRDIhP 46 T.vonseri. DPFGASTMORPWDRFSDPMDIAKARVEAAFEFFEKLDVPFFCFDRDIAP 106

T.saccharolytlcum. EGDTLRETNKNLDTIVAMIKDYLKTSKTKVLWGTANLFSNPRHGASTS 157 Thermdanaerobacterlum EGDTLRKTNKNLDTIVAMIKDYLKTSKTKVLWGTANLFSNPRHATS 157

C.bel]er~nckll EGKTLQETNKNLI T.marltima. EGKTLRETNKILI T.neapo1ltan.a. EGKTLRETNKILDKWERIKERMKDSNVKLLWGTANLFSHPRYHHGTT 157

DGKTLTETNKNLDEIVELAKKLQEETNIKPLUGTAQLFUHPRYMHGAATS 200 DGKTLAETNANLDEIVELAKQLQSETNIKPLWGTAQLEnHPRYMHGAATS 200 DGTTLEESNKNLDEVIELAKELQKGSKIKPLWGTAQLFLHPRYMHGGATS 198 .. . ... . f *..... . .*.._*.,~.: . . .. *. : : * _ . . . . ..

CNADVFAYAAAQIRKAIEITKELDGENYVFWGGREGYETLLNTGLELD 207 CSADVFAYAAAOVKXALEITKEIGGEGWFWGGREGYETLLNTDLGLELE 207

tertiary structures. The aligned sequences however revealed two signature sequences,

VXW(GP)GREG(YSTA)E and (LIVM)EPKPX(EQ)P, that were also recognized to be

present in the plant XI but in no other proteins in the SWISS-PROT (version 23) analysis.

It was strategized therefore to amplify a DNA fragment internal to the xylA gene of

Cereus pterogonus and Opuntia vulgaris and use it as a probe to screen a cDNA library

that is to be generated for the purpose. For a primer design for use in PCR technique, the

amino acid sequences of xylose isomerase available in GENBANK for the mesophilic

plants (Arabidopsis thaliana, Oryza saliva. Hordeum vulgare) and the therrnophilic

bacteria (Thermotoga maritime. Thermotoga neapolitana . Clostridium beijerincki NCIMB

8052, Thermoanaerobacter ethanolicus, Thermoanaerobacter yonseii,

l'hermoanaerobacter saccharolyticum , Thermoanaerobacterium sp.. Thermoanaerobacter

thermosulfurogenes) were aligned (Fig. 56) to determine the highly conserved regions. The

following sequences, FSVAFWHTF (position 86 to 93), CFHDRDIAP (position 141 to

150). EPTKHYD (position 279 to 288) and YVFGGRE (position 227 to 236) in the

Hordeum vulgare were found to be conserved among the 11 species compared. Two sets of

primers (BMBFIIRI and BMBF21R2) directed to the conserved regions were therefore

designed. The primer BMBFl and BMBRl was expected to yield a PCR product having

579bp and the primer pair BMBF2 and BMBR2, was expected to yield a PCR product that

was 258bp. The GcC content and the melting temperature (Tm) for these primers are given

under 'Methods'. Additionally a third set of primers was also designed to serve as a

degenerate pair of primers (BMBFjIRp).

Molecular Biology studies

Preliminary studies directed at determining the eukaryotic thermophilic xylose

i som~ase gene size of Cereus pterogonus and Opuntia vulgaris species employed the

~ ~ l y m e r a s e chain reaction technique as described under 'Methods'. The xerophytic

genomic DNA as well as the poly A' RNA was both employed as PCR templates in

independent attempts for the amplification process. While the former was expected to yield

an amplified product based on the DNA template sequence, the use of poly A+ RNA, for

the initial reverse transcription process, was expected to yield a cDNA-PCR product

devoid of the intron sequences and therefore of a lower molecular size than the DNA-PCR

amplified product.

Employing Cereus plerogonus and Opuntia vulgaris cDNA as template, the

BMBFzIRz primer pair yielded a single band of the expected size reproducibly. Genomic

DNA as the template did not however yield the desired amplified product. Using

BMBFIRI primer pair, PCR products of 600bp and 350bp were generated for Opuntia

vulgaris cDNA and a 250bp PCR product was observed for the Cereus pterogonus cDNA

(Plate 8).

However, BMBFIRI and BMBFzRz primers were not able to amplify the

genomic DNA XI segment. Degenerate primers were therefore designed and employed for

this purpose. The primer pair (BMBFdR3) was designed based on XI gene sequence

available in the GENBANK, since no information on thermophilic plants XI gene

sequences exist in the database. The degenerate primer pair yielded a PCR amplified

product of 800bp and 450bp bands when Cereus pterogonus genomic DNA was used as

template and a 600bp and 200bp PCR band when the Opuntia vulgaris genomic DNA was

used as template for the amplification process (Plate 9) Due to the limited availability of

prepared cDNA for further work, PCR amplification process employing the primer pair

BMBF,/R, wuld not be carried out, compounded by time limitations. Nonetheless,

nucleotide sequence determination of each of the PCR amplified product was attempted by

providing samples to Macrogen, South Korea and Bangalore Genei, India. Results of

sequencing obtained from both of these technical services did not indicate homology with

PLATE 8

Cereus pterogonus and Opuntia vulgaris genomic DNA and cDNA-PCR amplified products for xylose isomerase gene using of BMBFI/RI (A) and BMBFt/R2 (B) primers.

A B

1 2 3 4 5 6 7 8 9 1 0

Lane 1 - lOObp Lambda-DNA ladder as Molecular weight marker. Lane 2 - Cereus prerogonus DNA-PCR with primer BMBFllRl Lane 3 - Opunria ~~ i lgar i s DNA-PCR with primer BMBFJRI Lane 4 - Cereusprerogonus cDNA-PCR with primer BMBF!/R, Lane 5 - Opunriu i~ulguris cDNA-PCR with primer BMBFIRI Lane 6 - Cereus prerogonus DNA-PCR with primer BMBF2lR2 Lane 7 - Opuntia ~~ulgaris DNA-PCR with primer BMBF2/R2 Lane 8 - Cereus prerogonus cDNA-PCR with primer BMBF2IR2 Lane 9 - Opuntia t,ulgaris cDNA-PCR with primer BMBF21R2 Lane 10 - IOObp Lambda-DNA ladder as Molecular weight marker.

PLATE 9

PCR amplified product of BMBF.f13 primer for xylose isomerase gene of Cereus pterogonus and Opuntia vulgaris.

Lane 1 - IOObp Lambda DNA ladder as Molecular weight marker.

Lane 2 - Cereus prerogonus DNA-PCR with primer BMBFdR3

Lane 3 - Opirntia 19ulgaris DNA-PCR with primer BMBF?/RI

any of the known sequences of XI in the database, as determined through BLAST searches.

Hence, the nucleotide sequence data is not reported in this work.

Available information on xylose isomerase genes from several species (mesophiiic

plants, bacteria and thermophilic bacteria) indicate extensive molecular divergence in the

XI gene and protein sequences. The inability to generate a full length cDNA-PCR or DNA-

PCR amplified product will have to be considered against this background. More extensive

approaches employing additional primer designs, Southern blot and hybridization

techniques have to be carried out along with successive sum of the PCR technique to

establish the true nature of the thennophilic XI gene and its sequence. The use of "(P)

labeled primer probes and 1 or fluorescence labeled Digoxin probes will be required for

establishing the eukaryotic thermophilic XI gene identity. Work is therefore under progress

in this direction