210 Chiang Mai J. Sci. 2011; 38(2)

Chiang Mai J. Sci. 2011; 38(2) : 210-222www.science.cmu.ac.th/journal-science/josci.htmlContributed Paper

An Orphan Crop (Cucumeropsis mannii Naudin) withInteresting Phosphatase, βββββ-galactosidase andααααα-mannosidase Activities: Nutritional Approachand Biochemical CharacterizationDjary M. Koffi [a], Betty M. Faulet [b], Jean T. Gonnety [b], Mica l E. B dikou [a],Iri A. Zoro Bi [c], and S bastien L. Niamk *[a][a]Laboratoire de Biotechnologies, Fili re Biochimie-Microbiologie de l’Unit de Formation et de Recherche

Biosciences de l’Universit de Cocody, 22 BP 582 Abidjan, Co^ te d’Ivoire.[b]Laboratoire de Biochimie et Technologie des Aliments de l’Unit de Formation et de Recherche des

Sciences et Technologies des Aliments de l’Universit d’Abobo-Adjam , 02 BP 801 Abidjan, Co^ te d’Ivoire.[c] Laboratoire de G n tique de l’Unit de Formation et de Recherche des Sciences de la Nature del’Universit d’Abobo-Adjam , 02 BP 801 Abidjan, Co^ te d’Ivoire.*Author for correspondence; e-mail: niamkes@yahoo.fr

Received: 6 April 2010Accepted: 27 February 2011

ABSTRACTSeeds extract of Cucumeropsis mannii Naudin (Cucurbitaceae), an orphan and under-

researched crop was screened for enzymatic hydrolytic activities over a variety of natural andsynthetic substrates. Enzymatic activities noticed mainly consist of phosphatase (0.66 0.02UI/mg), β-galactosidase (0.26 0.03 UI/mg) and α-mannosidase (0.19 0.02 UI/mg) activities.The higher phosphatase activity observed suggest that this edible seed constitutes a potentialsource of phosphorus for human and animal diet. Exploration of biochemical properties hasrevealed that phosphatase, β-galactosidase and α-mannosidase activities were maximal at 45oC(pH 5.6), 55oC (pH 4.6) and 60oC (pH 5.0), respectively. The three enzymatic activities displayedalso interesting characteristics as relative pH stability, resistance to (or activation by) detergentsand hydrolytic specificity over natural substrates such as ATP, ADP, lactose and mannobioses.So, these disclosed enzymatic activities should deserve main investigations for furtherbiotechnological applications.

Keywords: Orphan crop, Cucumeropsis mannii seed, cucurbitaceae, phosphatase, β-galactosidase,α-mannosidase.

1. INTRODUCTIONThe usage of plant resources for various

biotechnological purposes as food industry,genetic and medicine is of economic importance,social value and essential for humanity survival.In this respect, it is imperative to collect andconserve representative stocks of emerging

plants with improved properties. It was fromthis perspective that neglected and underutilizedcrops also called minor or orphan crops [1, 2]such as those of the cucurbits family, haveattracted attention for scientific research.

Cucurbitaceae (Cucurbits) is a plant family

Chiang Mai J. Sci. 2011; 38(2) 211

commonly known as melons or gourds andincludes crops like cucumbers, squashes(including pumpkins), luffas, and watermelons.The family is predominantly distributedaround the tropics, where those with edible-seeded were among the earliest cultivatedplants in both the Old and New Worlds [3].These under-researched crops contribute tothe diet of a large scale portion of resource-poor consumers and at the same time generateincome for small-holder farmers in developingcountries, particularly in Africa [4]. Thus, it isa major family for economically importantspecies and some have nutritional, medicinaland other useable properties [5, 6]. In addition,they perform better than major crops of theworld under extreme soil and climaticconditions [4].

Cucumeropsis mannii Naudin, also knownas Mann’s Cucumeropsis and white-seed melonin English, is a member of the indigenousedible cucurbits [2]. This neglected andunderutilized vegetable has, at present, arousedattention due to its agronomic and economicpotentials. Indeed, it has become material forproduction improvement and can be used infood conserve [4]. These interesting potentialitieshave resulted from this cucurbit’s scientificinterest that had consisted in its extensivecharacterization.

In Côte d’Ivoire, five species of cucurbitsincluding Cucumeropsis mannii are very prizedfor their oleaginous seeds consumed astraditional soup thickeners locally calledpistachio soup, and also called egusi soup inCameroon, Nigeria and Benin [7]. Thisspecies of cucurbits is particularly reportedto be highly rich in nutrients [8], namelyprotein (36 2.17%) and fat (45.89 4.73%)and is thus becoming more and more studied.

The genetic, nutritional and agronomicpotentials of C mannii have been previouslyreported [6, 9-11], but the enzymatic potentialof their seeds has not been yet explored.

However, it is currently used as enzymes inbiotechnological process [12-14]. So, the presentwork is conducing to highlight some enzymaticcontents of Cucumeropsis mannii seeds with theaim of discussing their nutritional functionsand then, biochemically characterizing thebest ones for further biotechnologicalapplications.

2. MATERIALS AND METHODS2.1 Enzymatic Source and EnzymesExtraction

C. mannii seeds were obtained from thecollection of the University of Abobo-Adjam(Abidjan, Côte d’Ivoire). To obtain sufficientnumber of seeds, C. mannii was grown duringits appropriate cropping season in 2008 at theexperimental farm of the University (5o23latitude North, 4o00 longitude West, and 7 maltitude). After harvest, the fruits were splitusing a stainless steel machete, and then seedswere treated for the crude extract preparation.Cucurbit seeds (10 g) were ground using ablender in 20 ml sodium chloride solution0.9% (w/v). The homogenate was subjectedto sonication using a TRANSSONIC T420 for10 min, and then centrifuged at 10,000g for30 min. The supernatant filtered throughcotton was used as the crude extract (seedsextract).

2.2 Chemicalspara-nitrophenyl- (pNP-) glycopyranoside

substrates (pNP-α-D-mannopyranoside(pNP-α-D-Man), pNP-α-D-glucopyranoside(pNP-α-D-Glu), pNP-β-D-glucopyranoside(pNP-β-D-Glu), pNP-α-D-galactopyranoside(pNP-α-D-Gal), pNP-β-D-galactopyranoside(pNP-β-D-Gal), pNP-α-L-fucopyranoside(pNP-α-L-Fuc), pNP-α-L-arabinopyranoside(pNP-α-L-Ara), pNP-β-D-xylopyranoside(pNP-β-D-Xyl)), para-nitrophenyl phosphate(pNPP), phenyl phosphate, sodium pyrophos-phate, glucose-1-phosphate, glucose-6-

212 Chiang Mai J. Sci. 2011; 38(2)

phosphate, fructose-6-phosphate, fructose-1-phosphate, α-nicotinamide adenosinedinucleotide (α-NAD), adenosine-2-3’-cyclo-monophosphate, adenosine-5’-triphosphate(ATP), phytate, para-nitrophenol (pNP),sucrose, starch, carboxymethylcellulose(CMC), Inulin, xylan, lactose, 2-O-α-D-mannopyranosyl-D-mannopyranoside (α-1,2Mannobiose), 3-O-α-D-mannopyranosyl-D-mannopyranoside (α-1,3 Mannobiose), 6-O-α-D-mannopyranosyl-D-mannopyranoside(α-1,6 Mannobiose), D-mannose and D-glucose were purchased from Sigma Aldrich.Bovine serum albumin (BSA) was from FlukaBiochemika.

2.3 Hydrolytic Screening of Synthetic andNatural Substrates by Seeds Extract

For synthetic pNP-glycoside and pNP-phosphate hydrolytic activities, the crudeextract was mixed in a total volume of 250 μlcomposed of the aryl-glycoside or aryl-phosphate substrate (5 mM, startingconcentration) in 100 mM sodium acetatebuffer pH 5.0 and the crude extract (50 μlwhich correspond to 0.3 mg of protein).The reaction mixture was incubatedwith shaking at 37oC for 10 min. The liberatedpara-nitrophenol (pNP) was quantifiedspectrophotometrically at 410 nm underalkaline conditions (2% w/v, Na2CO3) referredto a standard pNP (absorbance as a functionof concentration) curve obtained in similarconditions. For natural substrates hydrolysis,the total volume was 300 μl, composed of50 μl of the crude extract and 125 μl ofsubstrate (0.25% w/v, final concentration) in100 mM sodium acetate buffer pH 5.0. Thereaction mixture was incubated with shakingat 37oC for 30 min, and the reactions werestopped by adding 150 μl of dinitro salicylicacid (DNS) and by heating the resultedsolution at 100oC for 5 min [15]. The liberatedreducing sugars were quantified spectrophoto-

metrically at 540 nm referred to a standardglucose (absorbance as a function of concen-tration) curve obtained in similar conditions.All values were determined in triplicate. Oneunit of enzymatic activities in the two cases(synthetic and natural substrates) released 1μmol of liberated product (pNP or reducingsugar) per min under the above conditions,respectively. The specific activity was expressedas μmol per min (UI) per mg of proteins.

2.4 Estimation of Protein ConcentrationThe concentration of the proteins was

measured using the Folin ciocalteu method[16]. Bovine serum albumin (BSA) was usedas the standard protein.

2.5 pH and Temperature OptimaThe effect of pH on the enzymatic

activities was determined by performing thehydrolysis of para-nitrophenylphosphate, para-nitrophenyl-β-D-galactopyranoside and para-nitrophenyl-α-D-Mannopyranoside in a seriesof buffers (100 mM) at various pH valuesranging from 3.0 to 6.2. The buffers usedwere sodium acetate buffer from pH 3.6to 5.5 and sodium citrate buffer from pH 3.0to 6.2. The pH values of each buffer weredetermined at 25oC. The effect of temperatureon phosphatase, β-galactosidase and αmannosidase activities was performed in 100mM acetate buffer (appropriate pHs) over atemperature range of 30 to 80oC by usingpNPP, pNP-β-D-Gal or pNP-α-D-Man(5 mM) as substrates under the enzyme assayconditions.

2.6 pH and Temperature StabilitiesThe pH stability of each enzymatic activity

was studied in a pH range of 3.0 to 6.2 with100 mM sodium citrate buffer. The bufferwas the same as that in pH and temperatureoptima study (above). After 2 h preincubationat 25oC, residual activities were measured at

Chiang Mai J. Sci. 2011; 38(2) 213

37oC for 10 min by adding substrates (pNPP,pNP-β-D-Gal and pNP-α-D-Man). Thethermal inactivation was determined at 37oCand at each enzymatic activity optimumtemperature. Enzymes in appropriate buffers(pHs) were exposed to each temperature forthe time range of 0-100 min. Aliquots werewithdrawn at intervals and immediatelycooled. Concerning thermal denaturation tests,aliquots of the crude extract were preheatedat different temperatures ranging from 30to 80oC for 15 min. Residual activities,determined in the three cases at 37oC underthe enzymes assay conditions, were expressedas percentage activity of zero-time control ofuntreated enzymes.

2.7 Effect of Some Chemical AgentsTo determine the effect of various

compounds (cations, chelating, sulphidrylspecific and reducing agents and detergents)as possible activators or inhibitors of theenzymatic activities, the crude extract waspreincubated at 37oC for 30 min with thecompounds and then, the activity wasassayed under the enzymes assay conditions.The residual activities were expressedas percentage refers to a control withoutchemical agents.

2.8 Characterization of HydrolyticSpecificity

The hydrolytic specificity was determinedby incubating the fraction of the seed extract(50 μl which correspond to 0.3 mg ofproteins) with appropriate substrates fordifferent times at 37oC in 100 mM sodiumacetate buffer (each optimum pH). For phos-phorylated substrates hydrolytic specificity,the inorganic phosphate (Pi) produced by thephosphatase activity was quantified by themethod of Heinonen and Lahti [17]. Glucoseand galactose released from the hydrolysis oflactose (1 mM) and mannose released from

differently linked mannobioses (1 mM) werevisualized through TLC plates. 3 μl Sampleswere spotted for each mixture. TLC plateswere run with butanol-acetic acid-water(9:3.75:2.25, v/v/v), and then developed withnaphto-resorcinol in ethanol and H2SO4 20%(v/v). The sugar spots were visualized at110oC for 5 min.

3. RESULTS3.1 Screening of Glycosidase and Phos-phatase Hydrolytic Activities

The potential of synthetic and naturalderivatives to act as substrates for the seedextract of Cucumeropsis mannii was assayed(Figure 1). We noticed that the crude extracthydrolyzed eleven (11) of the fourteen (14)substrates assayed. The best hydrolytic activitiesobserved were phosphatase activity (0.660.02 UI/mg) followed by β-D-galactosidase(0.26 0.03 UI/mg) and α-D-mannosidase(0.19 0.02 UI/mg) activities. There areapparently no α-D-glucosidase, α-D-fucosidaseand β-D-xylosidase activities (Figure 1).

3.2 pH and Temperature DependencesPhosphatase, β-galactosidase and α-

mannosidase activities were analyzed for theirhydrolytic pH and temperature dependences.The three enzyme activities were maximal at45, 55 and 60oC and at pHs 5.6, 4.6 and 5.0,respectively (Figures 2A and 3). They retained80% of their activity in sodium acetate bufferat a pH range of 4.0 to 6.2 (Figure 2B). Thethermal stability study has revealed that at37oC, the three activities remained stable for100 min (Figure 4A). However, at theiroptimum temperature, these activities wereless stable showing half-life (50% of activity) atleast at about 60 min (Figure 4A). Phosphatase,β-galactosidase and α-mannosidase conserved100% of their hydrolytic activity to temperaturesup to their optima. Above, their activitiesdeclined progressively as the temperature

214 Chiang Mai J. Sci. 2011; 38(2)

Figure 1. Screening of the seed extract from cucumeropsis mannii for several glycosidase andphosphatase activities over synthetic and natural substrates.

Substrates

Spec

ific a

ctiv

ity(1

0-1

μμμμ μmol

/min

/mg)

Figure 2. Effect of pH on phosphatase, β-galactosidase and α-mannosidase activities fromthe seed extract of Cucumeropsis mannii. (A) pH optimum, (B) pH stability.

pH

pH

Rel

ativ

e act

ivity

(%)

Res

idua

l act

ivity

(%)

Chiang Mai J. Sci. 2011; 38(2) 215

increased (Figure 4B). Values of temperaturecoefficients (Q10 ) calculated for the threeenzyme activities were found to be for around2; 1.8 and 1.5, respectively. From Arrhenius

plot, values of 66.8, 49.2 and 30.2 kJ/mol wereobtained for the activation energy of phos-phatase, β-galactosidase and α-mannosidaseactivities, respectively (Data not shown).

Figure 3. Effect of temperature on phosphatase, β-galactosidase and α-mannosidase activitiesfrom the seed extract of Cucumeropsis mannii.

Temperature (oC)

Rel

ativ

e ac

tivi

ty (

%)

Figure 4. Thermal stability of phosphatase, β-galactosidase and α-mannosidase activities fromthe seed extract of Cucumeropsis mannii. (A) Thermal inactivation, (B) Thermal denaturation.

Time (min)

Res

idua

l ac

tivi

ty (

%)

Temperature (oC)

Res

idua

l ac

tivi

ty (

%)

216 Chiang Mai J. Sci. 2011; 38(2)

Figure 5. Effect of cations, chelating agent and urea on phosphatase, β galactosidase andα mannosidase activities from the seed extract of Cucumeropsis mannii.

Res

idua

l ac

tivi

ty (

%)

Chemicals

Figure 6. Effect pCMB, L-cysteine and â-mercaptoethanol on phosphatase β-galactosidaseand α-mannosidase activities from the seed extract of Cucumeropsis mannii.

Res

idua

l ac

tivi

ty (

%)

Chemicals

3.3 Effect of Cations, Chelating, SulphidrylSpecific and Reducing Agents

The effect of several chemicals on phos-phatase, β-galactosidase and α-mannosidaseactivities by using the appropriate substrate wasstudied (Figures 5 and 6). All chemicals tested(Na+, Ca2+, Ba2+, K+, Mg2+, EDTA, urea,pCMB, L-cysteine and β-mercaptoethanol)were found to be without significant effecton phosphatase activity (residual activity in therange of 93.5 2.1 to 109.2 3.5%). As

concerned β-galactosidase activity, it wasslightly inhibited for around 23% by pCMB(5 mM). However, this activity was enhancedin a range of 113 2.1 to 126.7 2.6% by5 mM or 1% final concentration of Ca2+, Ba2+,EDTA, L-cysteine and β-mercaptoethanol(Figures 5 and 6). For α-mannosidase activity,most chemicals had no effect. Only Ba2+,EDTA and urea have shown to be relativelyinhibitory (between 22 and 42% of inhibition)(Figure 5).

Chiang Mai J. Sci. 2011; 38(2) 217

3.5 Substrate Hydrolytic SpecificityPhosphatase, β-galactosidase and α-

mannosidase activities from seeds extract ofCucumeropsis mannii were assayed for theircapabilities to hydrolyze different specificsubstrates (Table 2). The results showed thatphosphatase activity hydrolyzed a broad rangeof phosphorylated substrates. The highestactivity was observed with p-nitrophenylphosphate (100 2.1%) followed byadenosine-5’-triphosphate (83.2 3.3%) andphenyl phosphate (72.5 2.0%). However, thehydrolysis of the two latter substratesremained interesting compared to that ofsodium phytate (12.4 1.8%) and α-

nicotinamide adenine dinucleotide (11.50.9%) which were less specific (Table 2). Theability of β-galactosidase activity from seedsextract of C. mannii to greatly hydrolyzelactose, a natural substrate, has been showed(Figure 7A). Thin layer chromatography(TLC) analysis of the hydrolysis of naturalsubstrates namely α-1,2; α-1,3 and α-1,6mannobioses by the α-mannosidase activityshows that, the three differently linkeddisaccharides were cleaved at different rates.α-mannosidase hydrolytic activity on α-1,2mannobiose seemed to be greater than thaton the two other linkages (Figure 7B).

3.4 Effect of DetergentsCationic, non-ionic and anionic detergents

currently used for protein denaturing haveshown, by and large, no significant effecton the three enzyme activities. However,tetradecyl trimethyl ammonium bromide,hexadecyl trimethyl ammonium bromide,polyoxyethylene-9-lauryl ether and polyxy-

ethylene-10-tridecyl ether were found toenhance for up to 152 5.1% β-galactosidaseactivity while sodium dodecyl sulphatedisplayed a strong inhibitory effect (foraround 96% inhibition) on phosphatase, β-galactosidase and α-mannosidase activities(Table 1).

Relative activity (%)Detergents*

Phosphatase βββββ-Galactosidase ααααα-Mannosidase

Control 100 100 100

Cationic

Tetradecyl Trimethyl Ammonium Bromide 75.5 4.8 152.0 5.7 85.7 3.6Hexadecyl Trimethyl Ammonium Bromide 80.9 3.7 143.0 5.2 86.1 4.3

Non-ionic

Tween 80 92.4 4.6 94.0 4.3 91.4 5.1Lubrol Wx 107.4 3.8 100.0 2.4 93.1 2.5Triton X-100 101.1 5.6 108.0 3.3 94.3 5.3

Anionic

Polyoxyethylene-9-lauryl ether 107.4 4.3 122.0 5.2 93.0 4.3Polyxyethylene-10-tridecyl ether 100.0 2.2 119.0 4.5 102.2 5.3Polyxyethylene-10-oleyl ether 105.3 2.5 102.0 5.3 84.4 2.5Sodium dodecyl sulphate 11.6 4.4 41.7 3.9 5.3 1.9

* = Assays were performed at 37 oC for 10 min with 1% (w/v) starting concentration of detergent.

Table 1. Effect of the detergents on phosphatase, β-galactosidase and α-mannosidase activitiesfrom the seed extract of cucumeropsis mannii.

218 Chiang Mai J. Sci. 2011; 38(2)

Table 2. Hydrolytic specificity of phosphatase activity from the seed extract of cucumeropsismannii over some phosphorylated substrates.

Substrates* Hydrolytic activity (%)

p-Nitrophenyl phosphate 100.0 2.1Adenosine-5’-triphosphate (ATP) 83.2 3.3Phenyl phosphate 72.5 2.0Sodium pyrophosphate 50.0 2.4Glucose -6-phosphate 41.2 1.4Fructose-6-phosphate 32.1 1.3Glucose -1-phosphate 24.4 1.2Fructose-1-phosphate 23.0 2.1Adenosine-2’3’-cyclomonophosphate 19.8 3.2Sodium phytate** 12.4 1.8α-Nicotinamide adenine dinucleotide 11.5 0.9

* = Assays were performed at 37 oC for 30 min with 5 mM final concentration of substrate,** = This reaction was performed at 50C for 2 h.

Figure 7. TLC plates showing β-galactosidase and α-mannosidase activities from the seedextract of Cucumeropsis mannii towards differently linked disaccharides. (A) β-galactosidase activityon lactose. Lane 1, glucose; Lane 2, galactose; Lane 3, lactose; Lane 4, reaction mixture. (B)α mannosidase activity on α-1,2; α-1,3 and α-1,6-mannobioses. Lane 1, crude extract; Lane 2,mannose; Lane 3, mannobiose; Lanes 4-6, reaction mixtures with α-1,2; α-1,3 andα-1,6-mannobiose, respectively.

(A) (B)

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4. DISCUSSIONEnzymes are essential biocatalysts to

living organisms. Indeed, different aspects ofbiological functions have been resolved bystudying physiological enzymes [18]. With theaim of diversifying sources of enzymes forfurther biotechnological application, thepractical approach in enzymology to explorenew biocatalysts, namely screening have beenconduced. This study led to the assertion thatC. mannii is an excellent source of enzymes,particularly an interesting phosphatase, β-galactosidase and α-mannosidase sources.Nevertheless, phosphatase activity wasnumerically large than the two latter.

Higher phosphatase activity is a goodindicator of the present phosphorus status ofa plant [19]. In this respect, it may be suggestedthat C. mannii seeds represent an importantsource of phosphorus for human and animaldiet. However, the increase in phosphataseactivity depended upon plant age, plant speciesand soil type [20]. Therefore, these affectingfactors should also be taken into accountfor main investigations. Phosphatase, β-galactosidase and α-mannosidase specificactivities noticed in the crude extract of C.Mannii seeds appeared to be higher thanthose already reported for plants and othersources, which were considered for purification[21-23]. So, this crop constitutes an interestingsource of phosphatase, β-galactosidase or α-mannosidase which deserves be explored ingreat details for specific bio-industrial uses.

Biochemical characterization of the threeenzymatic activities has revealed common andparticular properties compared to thosealready reported. Indeed, pH and temperaturestudies have shown that these activities areacidic and mesophilic. This behaviour is inaccordance with the majority of plant phos-phatase, β-galactosidase and α-mannosidaseactivities already reported [21, 24, 25]. Thethree activities were conserved for around

80% in a pH range of 4.0 to 6.2 for 2 h.This residual stability remains suitable, andthen constitutes a good compromise forperforming hydrolysis or synthesis reactionsfor up to 2 h.

The behaviour of C. mannii seed extractphosphatase and β-galactosidase hydrolyticactivities in the presence of divalent cationsand cations chelator (EDTA) is tempting tospeculate that the concerning ions were notstrictly essential for the two enzymatic activities.However, the sensitivity of β-galactosidasehydrolytic activity to pCMB led to assumethat -SH groups participate probably inthis enzymatic reaction. As concerned α-mannosidase activity, the strong inhibitoryeffect caused by EDTA may suggest itsmetallo-dependence’s as already shownin the case of Drosophila melanogaster α-mannosidase [26].

There is by and large, a tendency for thethree enzymatic activities to remain stable oractivated by most of detergents tested. Thisbehaviour should constitute an advantageouscharacteristic for their potential bio-industrialusage. Furthermore, the strong stimulatoryeffect displayed by the cationic detergentstested on β-galactosidase activity make themparticularly useful when extract this activity byimproving its stability for further specificcharacterization.

Phosphatase activity from the seed extractof C. mannii was not strictly specific since itreleased inorganic phosphate from a varietyof phosphorylated substrates. Although thisactivity was maximal toward the synthetic arylsubstrate p-nitrophenyl phosphate, it alsoshowed considerable activity other a naturalsubstrate (ATP). This relatively high activitytoward ATP indicates the possibility to applythis enzymatic activity to synchronous enzyme-reaction system, which needs energy resultingfrom the hydrolysis of this substrate [24]. Aswell as phosphatase activity, β-galactosidase

220 Chiang Mai J. Sci. 2011; 38(2)

activity has also showed ability to greatlyhydrolyze a natural substrate (lactose) andthus, could serve as an exo-glycosidase inreleasing galactose from oligogalactosides.Furthermore, enzymatic hydrolysis of lactosehas two main biotechnological applications:the utilization of whey as glucose and galactose(the hydrolysates) having greater fermentationpotential [27] and in the production of lowlactose milk (and dairy products made from it)for consumption by lactose intolerant persons.To date, the majority of industrially used β-galactosidase preparations were either frombacterial or fungal sources [28]. However,Dey [29] had reported that widely distributedplant β-galactosidase could be a goodsubstitute for lactose hydrolysis in industriesdue to easy availability and cost effectiveness.In this respect, β-galactosidase from C. Manniiseeds could also be explored for industrialapplication. As for α-mannosidase activity, itdisplayed broad natural substrate specificityby hydrolyzing α-1,2; α-1,3 and α-1,6mannobioses linkages. This behaviour bearsout a close resemblance to α-mannosidasesinvolved in the degradation of oligomannosetype free N-glycans which exhibit biologicalactivity as growth factor during the earlydevelopment of plant [30] and stimulation offruit ripening [22, 31]. For the specificapplication, the purified α-mannosidase(s)from the seeds extract of Cucumeropsis manniicould also find the importance in thecombination with xylanase in pulp andpaper industry as already showed by Viikariet al. [32].

5. CONCLUSIONTo sum up this report, it may be conclude

that phosphatase, β-galactosidase and αmannosidase activities from the seed extractof Cucumeropsis mannii displayed interestingnutritional and biochemical characteristicsthat deserve main investigations for further

biotechnological applications.

ACKNOWLEDGEMENTSThis work was supported by a PhD grant

of the first author. The authors are grateful toLaboratoire de G n tique (UFR des Sciencesde la Nature, Universit d’Abobo-Adjam ,Cô te d’Ivoire) for technical assistance.

REFERENCES

[1] IPGRI., Neglected and underutilized plantspecies: strategic action plan of the InternationalPlant Genetic Resources Institute (IPGRI).Rome: IPGRI, 2002.

[2] Rasul M.G., Hiramatsu M. and OkuboH., Genetic relatedness (diversity) andcultivar identification by randomlyamplified polymorphic DNA (RAPD)markers in teasle gourd (Momordica dioicaRoxb). Sci. Hortic., 2007; 111: 271-279.

[3] Sanjur O.I., Piperno D.R., Andres T.C.and Wessel-Beaver L., Phylogeneticrelationships among domesticated andwild species of Cucurbita (Cucurbitaceae)inferred from a mitochondrial gene:implications for crop plant evolution andareas of origin. Proc. Nat. Acad. Sci. USA.,2002; 99: 535-540.

[4] Esfeld K., Plaza S. and Tadele Z., Bringinghigh-throughput techniques to orphan crop ofAfrica: Highlights from the Tef TILLINGProject. Tef Biotechnology project,Institute of Plant Sciences, University ofBern, Switzerland, 2009.

[5] Bates M.D., Robinson R.E. and JeffereyC., Biology and Utilization of the Cucurbitaceae.Ithaca and London, 1990.

[6] Loukou A.L., Gnakri D., Dj Y., KippreA.V., Malice M.J.P.B. and Zoro B, I.A.,Macronutrient composition of threecucurbit species cultivated for seedconsumption in Cô te d’Ivoire. Afr. J.Biotechnol., 2007; 6: 529-533.

Chiang Mai J. Sci. 2011; 38(2) 221

[7] Zoro Bi I., Koffi K.K., Dj Y., MaliceM., Baudoin J.P. and Baus E.,. Biodiversityof cucurbits consumed as sauce thickenerin Cô te d’Ivoire: a capital resource forthe economic prosperity of rural women;in Segers H. and Desmet P., eds., TropicalBiodiversity: Science, Data, Conservation,Global Biodiversity Information Facility(GBIF) Brussels (Belgium), 2005: 158-167.

[8] Enujiugha V.N. and Ayodele-Oni O.,Evaluation of nutrients and some anti-nutrients in lesser-known, underutilizedoilseeds. Int. J. Food Sci. Technol., 2003; 38:525-528.

[9] Zoro Bi I., Koffi K.K., Dj Y., MaliceM. and Baudoin J.P., Indigenous cucurbitsof Cô te d’Ivoire: a review of their geneticresources. Sci. Nat., 2006; 3: 1-9.

[10] Osuji O.J., Okoli E.B. and Heslop-Harisson P.J.S., Cytology and molecularcytogenetics of Cucumeropsis manniiNaudin: implications for breeding andgermplasm characterization. Int. J. Bot. 2,2006; 2: 187-192.

[11] Achu M.B., Fokou E., Tchiegang C.,Fotso M. and Tchouanguep F.M.,Atherogenicity of Cucumeropsis mannii andCucumis sativus oils from Cameroon. Afr.J. Food Sci., 2008; 2: 021-025.

[12] Dissin K. and Uerkvitz W., Class Bnonspecific acid phosphatase fromSalmonella typhimurium LT2 Phospho-transferase activity, stability and thiolgroup reactivity. Enzyme Microb. Technol.,2006; 38: 683-688.

[13] Yapi D.Y.A., Niamk S.L. and KouamL.P., Biochemical characterization of astrictly specific beta-galactosidase fromthe digestive juice of the palm weevilRhynchophorus palmarum larvae. Entomol.Sci., 2007; 10: 343-352.

[14] B dikou E.M., Kon M.F., Ahi A.P.,Gonnety T.J., Faulet M.B., Kouam L.P.

and Niamk L.S. α-Mannosidases fromthe digestive fluid of Rhynchophoruspalmarum larvae as novel biocatalysts fortransmannosylation reactions. Appl.Biochem. Biotechnol., 2009; 162: 307-320.

[15] Bernfeld P., Amylases and proteases; inColowiiz S.P. and Kaplan N.O., eds.,Methods in Enzymology, Academic Press,New York, 1955: 149-158.

[16] Lowry O.H., Rosebrough N.J., Farrra L.and Randall R.J., Protein measurementwith Folin-phenol reagent. J. Biol. Chem.,1951; 193: 265-275.

[17] Heinonen J.K. and Lahti R.J., A new andconvenient colorimetric determination ofinorganic orthophosphate and itsapplication to the assay of inorganicpyrophosphate. Anal. Biochem., 1981; 113:313-317.

[18] Terra W.R. and Ferreira C., Insect digestiveenzymes: properties, compartmentaliza-tion and function. Comp. Biochem. Physiol.B., 1994; 109: 1-62.

[19] Mclachlan K.D., Leaf acid phosphataseactivity and the phosphorus status offield-grown wheat. Aust. J. Agric. Res.,1982; 33: 453-464.

[20] Tarafdar J.C. and Jungk A., Phosphataseactivity in the rhizophere and its relationto the depletion of soil organicphophorus. Biol. Fert. Soils., 1987; 3:199-204.

[21] Li S.C., Han J.W., Chen K.C. and ChenC.S., Purification and characterization ofisoforms of β-galactosidases in mungbean seedlings. Phytochemistry, 2001; 57:349-359.

[22] Ahi A.P., Gonnety T.J., Faulet B.M.,Kouam L.P. and Niamk S.L.,Biochemical characterization of twoα-mannosidases from breadfruit(Artocarpus communis) seeds. Afr. J. Biochem.Res., 2007; 1: 106-116.

222 Chiang Mai J. Sci. 2011; 38(2)

[23] Konan K.H., Ahi A.P., Kouadio N.E.J.P.,Gonnety T.J. and Kouam L.P. Charac-terization of purple acid phosphatasefrom breadfruit (Artocarpus communis)seed. J. Appl. Biosc., 2008; 10: 449-460.

[24] Gonnety J.T., Niamk S., Faulet B.M.,Kouadio E.J.P.N. and Kouam L.P..Purification and characterisation of threelow-molecular-weight acid phosphatasesfrom peanut (Arachis hypogaea) seedlings.Afr. J. Biotechnol., 2006; 5: 35-44.

[25] B dikou E.M., Ahi A.P., Kon M.F.,Gonnety T.J., Faulet M.B., Kouam L.P.and Niamk L.S., Biochemical propertiesof extracellular α-mannosidases from thedigestive fluid of Rhynchophorus palmarumlarvae. Bull. Insect., 2009; 62: 75-84.

[26] Van Den Elsen J.M., Kuntz D.A. andRose D.R., Structure of Golgi alpha-mannosidase II: a target for inhibition ofgrowth and metastasis of cancer cells.EMBO J., 2001; 20: 3008-3017.

[27] Kosaric N. and Asher Y.J., The utilizationof cheese whey and it components, inFiechter A., eds., Advances in BiochemicalEngineering, Springer, New York, 1985:25-60.

[28] Dwevedi A and Kayastha A.M., Optimalimmobilization of β-galactosidase fromPea (PsBGAL) onto Sephadex andchitosan beads using response surfacemethodology and its applications. Biores.Technol., 2009; 100: 2667-2675.

[29] Dey P.M., Biochemistry of the multipleforms of glycosidases in plants, in MeisterA., eds., Advances in Enzymology and RelatedAreas of Molecular Biology, John Wiley,New York, 1984: 180-186.

[30] Preim B., Morvan H., Hafez M.A. andMorvan C., Influence of a plant Glycanof the Oligosaccharide Types on theGrowth of flax Plantlets. CR Acad. Sci.Paris., 1992; 311: 411-416.

[31] Preim B. and Gross K.C., Mannoxyl andxylosyl containing glycans promotetomato (Lycopersicon esculentum M.). PlantPhysiol., 1992; 98: 399-401.

[32] Viikari L., Kantelinen A., Sundquist J. andLinko M., Xylanases in bleaching: froman idea to the industry. FEMS Microb.Rev., 1994; 13: 335-350.

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