Evaluation of Tinospora cordifolia Amylase as a Commercial Digestive Enzyme of Plant Origin

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Evaluation of Tinospora cordifoliaAmylase as a Commercial DigestiveEnzyme of Plant OriginAbhishek Mukherjee a , Subhasree Sengupta a , Lalita Ray b &Subhabrata Sengupta aa Department of Biotechnology , Heritage Institute of Technology,Anandapur , Kolkata , West Bengal , Indiab Department of Food Technology and Biochemical Engineering ,Jadavpur University , Kolkata , West Bengal , IndiaPublished online: 09 Mar 2012.

To cite this article: Abhishek Mukherjee , Subhasree Sengupta , Lalita Ray & Subhabrata Sengupta(2012) Evaluation of Tinospora cordifolia Amylase as a Commercial Digestive Enzyme of Plant Origin,Journal of Herbs, Spices & Medicinal Plants, 18:1, 58-76, DOI: 10.1080/10496475.2011.649515

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Journal of Herbs, Spices & Medicinal Plants, 18:58–76, 2012Copyright © Taylor & Francis Group, LLCISSN: 1049-6475 print/1540-3580 onlineDOI: 10.1080/10496475.2011.649515

Evaluation of Tinospora cordifoliaAmylase as a Commercial Digestive

Enzyme of Plant Origin

ABHISHEK MUKHERJEE,1 SUBHASREE SENGUPTA,1

LALITA RAY,2 and SUBHABRATA SENGUPTA1

1Department of Biotechnology, Heritage Institute of Technology,Anandapur, Kolkata, West Bengal, India

2Department of Food Technology and Biochemical Engineering,Jadavpur University, Kolkata, West Bengal, India

Tinospora cordifolia stem contained 470 + 50, 3.0 + 1.5, and 1.5+ 0.5 units of amylase, maltase, and isomaltase, respectively, pergram of fresh tissue. Amylase was more thermo- and acid-stablethan fungal, porcine pancreatic and human salivary amylasesand liberated much more reducing sugar and glucose from cerealstarches, amylopectin and glycogen than fungal enzyme. Crudeenzyme hydrolyzed maltose, isomaltose, raffinose, melezitose andraw starch and therefore does not require the participation ofintestinal disaccharidases for complete digestion of dietary starchinto glucose. The enzyme protein was nontoxic at an oral dose of1.5 g.Kg-1 body weight.

KEYWORDS maltase, cereal starch, amylopectin, sucrase-isomaltase

INTRODUCTION

Food and pharmaceutical industries prefer the use of plant enzymes in placeof microbial enzymes for various reasons (16). Digestive enzyme prepara-tions mostly contain plant proteases (bromelain, papain) commonly with

Received May 20, 2011.Address correspondence to Subhabrata Sengupta, Department of Biotechnology, Heritage

Institute of Technology, Anandapur, Chowbaga Road, Kolkata 700107, West Bengal, India.E-mail: [email protected]

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Digestive Enzyme Cocktail from Tinospora cordifolia 59

fungal amylase (Aspergillus oryzae). Large amounts of plant proteases andmalt amylase are consumed by brewing, food–processing, and pharmaceu-tical industries (9,29). Although the digestive enzyme from A. oryzae isconsidered to be safe for human health, there are many possible humanhealth hazards associated with the producer fungus itself (6). Additionally,the fungal amylase has also been reported to contain inhalation allergen(2). Plants as sources were reported to be of family I (extracellular), familyII (cytosolic), or family III (plastidal) amylases (38). A non-cereal, non-leguminous plant, Tinospora cordifolia (Meninspermaceae), was reportedto produce substantial amount of extracellular saccharifying amylase in theplant body (31). The plant is a succulent climbing shrub, indigenous toand found widely distributed in India. It grows wild on unconditioned soilthroughout the year and produces huge biomass in a short period. The trib-als of north Gujarat (India) use this plant in their daily life as food and/ormedicine (35). Indian traditional ayurvedic system of medicine prescribesaqueous extracts of this plant for treatment of diseases like diabetes, debil-ity, hepatitis, dyspepsia, and jaundice (34). Although a large number of plantamylases were purified and characterized (3,19,21), none was suitable forcommercial production (11). The present study evaluated the amylase fromT. cordifolia in terms of acid stability, cereal saccharification, cost, and safety.

MATERIALS AND METHODS

Materials

Crude fungal amylase (Aspergillus oryzae, A6211; crude lyophilized pow-der); pancreas acetone powder (porcine, P4251, source of crude pancreaticamylase); salivary amylase (A1031; lyophilized powder); glycogen (oys-ter shell); pullulan (from Aureobasidium pullulans); raffinose; melizitose;inulin; nigeran; maltose; isomaltose; 1-o-methyl-α-D-glucoside;soluble starch(potato); bovine serum albumine (BSA; fraction-V, A3059); dinitrosali-cylic acid (DNSA); and Bradford reagent were all purchased from SigmaChemicals, St. Louis, MO, USA; p-nitrophenyl-α-D-glucopyranoside was pur-chased from SRL, India. Trehalaose was a product of British Drug House(BDH), and; glucose oxidase-peroxidase (GOD-POD) reagent was a productof Span Diagnostics, India. Cysteine, calcium chloride and ethylene-diamine-tetra acetic acid-disodium salt (EDTA) were purchased from Merck, India.Amylopectin (potato) was purchased from Hi-Media. All other chemicalsused were of chemically pure grade.

Extraction of Enzyme

Crude enzyme was extracted from T. cordifolia as described previously (32).The process involved maceration of 400 g of washed small stem pieces

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60 A. Mukherjee et al.

of the plant (1–2 cm long) with 2 vol. (w/v) of 0.1 M acetate buffer pH5.0 containing 10 mM calcium chloride, 10 mM cysteine, 5 mM ethylenedi-aminetetraacetic acid (buffer A) at 50◦C and squeezing the paste through anylon cloth. The process was also performed on separate parts of the plant(stem bark, stem stick, leaves, and seeds). The extract obtained by blend-ing entire stem was processed further. Ethanol (15% v/v) was added to thegreenish extract, and the mixture was kept overnight at 4◦C and filtered toremove the precipitate that settled at the bottom. Enzyme protein was pre-cipitated out from the filtrate at –10 ◦C by further addition of 4 volumes ofchilled ethanol. The precipitate was collected by centrifugation at 4◦C anddissolved in buffer A. The solution was dialyzed against the same buffer andused as a source of crude enzyme.

Determination of Storage Stability of T. cordifoliaas a Source of Enzyme

For determination of the storage stability, the enzyme was prepared fromdifferent sources and stored in the following ways: (1) Washed fresh stem(200 g) were cut into 5- to 6-cm-long pieces, air–dried, and kept either atroom temperature (25◦ to 30◦C) or in cold (0◦ to 4◦C) in sealed polythenebags; (2) 1.2 kg of fresh plant was cut into small pieces as mentioned aboveand was dried by lyophilization. The dried mass (400 g) was stored sepa-rately at room temperature in airtight containers; (3) lyophilized plant stemswere crushed into fine powder and stored at room temperature; (4) 1.0 kgof fresh plant was cut into small pieces, dried in a hot air oven at 40◦Cfor 24 h, ground into fine powder, and stored at room temperature in air-tight containers; (5) and ethanol-precipitated enzyme (as mentioned earlier)was lyophilized, and dry enzyme preparation was stored at room tempera-ture. Plant materials and dry enzyme stored under all above conditions wereassayed for amylase activity at 3-month intervals during the 18 months ofstorage.

Determination of pH and Thermal Stability of CrudeAmylase Preparation

The pH optima of amylases from various sources were determined in the pHrange 3.0 to 7.0. The mixture (2 mL) containing 4.0 U of amylase preparedfrom different sources was incubated with 1% (w/v) soluble starch (potato)at 37◦C for 10 min. The enzyme activity was determined as described underAnalytical Procedures. The pH stability of enzyme was determined by incu-bating a mixture containing 10 U.mL−1 of amylase from different sources at37◦C for 1 h at different pH buffers (pH 3.0–7.0). Residual enzyme activ-ity was determined after dilution of the enzyme in optimum pH buffer

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(0.1M acetate buffer, pH 5.0 at 37◦C for fungal and plant amylase, and0.1 M phosphate buffer, pH 7.0 at 37◦C for pancreatic and salivary amylase).Acid stability (pH 3.0) of amylases from different sources was determinedas follows: enzyme solutions (20 U.mL−1) in 0.1M glycine-HCl buffer ofpH 3.0 were incubated at 37◦C; aliquots were withdrawn at different timeintervals; and the residual enzyme activities were determined using opti-mal buffers at temperature for each type of amylase as mentioned earlier.The optimum temperature for amylases from different sources was deter-mined in the temperature range from 30◦ to 80◦C. In the reaction mixture(2 mL), 0.5U of enzyme was incubated with 1% (w/v) soluble starch in req-uisite buffers at different temperatures for 15 min. Temperature stability wasdetermined in the range of 30◦ to 80◦C. The incubation mixtures containing10 U. mL mL−1 of amylase from different sources in requisite buffer solu-tions were pre-incubated at different temperatures for 30 min. Fixed aliquotswere withdrawn at different time intervals, and residual enzyme activitiesweredetermined.

Digestion of Various Cereals by Crude Enzyme

Corn flour, wheat flour, rice powder, and gram seed powder (purchased froma local market) were well gelatinized in water (4% w/v) at 100◦C for 5 min byconstant stirring. Digestion mixtures (10 mL) containing 2 % (w/v) gelatinizedcereal starches in 0.1M acetate buffer, pH 5.0, and 100 U of amylase (fromfungal and plant source) were incubated separately at 37◦C for 2 h. Reducingsugar and total glucose at different time intervals were estimated as describedearlier. The same methodology was followed for determination of hydrolysisof glycogen and amylopectin. Saccharification (%) was calculated accordingto the method of Gromada et al. (14).

Toxicity Test of the Crude Concentrated Enzyme Preparation

PREPARATION OF THE SAMPLE

Nine hundred g of fresh stem was collected, and enzyme was extracted asdescribed earlier. The enzyme solution was concentrated (by ultrafiltrationusing PM-10 membrane), dialyzed against distilled water, and lyophilized toget 14 g dry powder (containing 28,500 ± 500 U of amylase.g−1). This drypowder was administered orally to the animals in different doses.

ANIMALS

Healthy in-bred albino rats of Wister strain, aged between 80 and 95 daysand weighing 130 to 150 g, were used in this study. The rats were housedunder 12h/12h light-dark cycle in a temperature- and humidity-controlledenvironment with free access to food and water. The study was conducted

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in the animal house of Indian Institute of Chemical Biology, Kolkata, Indiafollowing institute ethical committee–cleared guidelines.

ACUTE TOXICITY TEST

An acute toxicity test was performed as described earlier (1). Six groupsof rats, each group containing six animals, were used in the present study.Crude lyophilized T. cordifolia enzyme preparation was administered orally(suspended in normal saline) to the five groups of albino Wister rats inthe doses of 0.20, 0.4, 0.8, and 1.5 g. kg−1 body weight, respectively. Thesixth group (control) was given equivolume of normal saline. Food waswithheld for 12 h before the experiment. Rats were continuously observedfor mortality (if any) and behavioral responses till the seventh day. Bodyweight and food intake were recorded daily.

SUBACUTE TOXICITY TEST

A subacute toxicity test was performed as given by Mythilpriya et al. (24).Four groups of rats, each containing eight animals (of either gender) wereused in the present study. Male and female rats were housed in separatecages. Crude lyophilized T. cordifolia enzyme preparation was administeredorally (suspended in normal saline) to the three groups in the doses of 0.1 g(5,700 U), 0.2 g (11,400 U), and 0.3g (17,100 U) kg−1 body weight twicedaily, respectively, for 20 days. The control group was given equivolume ofnormal saline. Body weight and food intake were recorded daily. Rats werecontinuously observed for mortality (if any) and behavioral responses till theforty-fifth day.

Analytical Procedures

Plant and fungal amylase activities were determined in 0.1M acetate buffer,pH 5.0 at 50◦C using 1 % (w/v) soluble starch (potato) as substrate. Reducingsugar formed was estimated by dinitro salicylic acid reagent method (33).Activities of salivary and pancreatic amylases were determined similarlyusing optimal buffers and temperature conditions. Enzyme activity (U) wasexpressed in terms of μ moles of maltose equivalent liberated per minunder the assay conditions. Pullulanse, 1-O-methyl-α-D-glucosidase, raf-finase, melizitase, and inulinase activities were determined in the sameway using 1% (w/v) each of pullulan, 1-O-methyl-α-D-glucoside, raffinose,melizitose, and inulin, respectively, as substrate. Maltase, isomaltase, treha-lase, and nigeranse activities were determined at pH 5.0 at 37◦C using 1%(w/v) each of maltose, isomaltose, and trehalose and 0.5% (w/v) nigeran,respectively, as substrates, and total glucose formed was estimated by glu-cose oxidase–peroxidase (GOD-POD) reagent method (5). Enzyme activity

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(U) was expressed in terms of μ mole of glucose liberated per min underthe assay conditions. Isoamylase activity was determined at pH 5.0 at 50◦Cusing glycogen as substrate according to the method of Spencer-Martins(37), determining the increase in iodine-staining power. Activity of theextract on p-nitrophenyl-α-D-glucopyranoside was determined by measur-ing the amount of p-nitro phenol released under the assay conditions.Dextrinizing activity of the enzyme was determined iodometrically at 37◦Cas described by Sengupta and Sengupta (30) using 2% (w/v) each of solu-ble starch, amylopectin, and crude gelatinized cornstarch as substrates andtaking α-amylase of Aspergillus oryzae as reference enzyme. Protein wasestimated using coomassie blue assay reagent (7). End product analysis wasdone by the thin-layer chromatography method (12).

RESULTS

Storage stability of the source and preparation of crude amyalse fromT.cordifolia The storage stability of enzyme in cut T. cordifolia stem asraw material was not very good. Recovery of amylase from plant stemsgradually lowered with the storage of fresh plant both at room tempera-ture and at 0◦ to 4◦C (Figure 1). Activity, above 50% of the original couldnot be recovered after 1 week of storage under both these conditions (seeFigure 1). However, plant stem pieces dried by lyophilization and kept atroom temperature in sealed containers retained more or less full enzyme

FIGURE 1 Storage stability of raw material stored at 0–4◦C: dry plant stem ( ), at roomtemperature (25–30 ◦C): dry plant stem ( ), Fresh extraction ( ), lyophilized stem pieces ( )and ethanol precipitated dry lyophilized enzyme ( ).

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activity over the 18–months’ storage (see Figure 1). Enzyme activity extractedin the aqueous medium remained stable in cold (4◦C) for more than 6 months(see Figure 1). T.cordifoila amylase was easily extractable from the plant, asa substantial amount of enzyme was simply leached out from the cut chipswhen stirred in the buffer, preferably at 50◦C, but the yield was poor whenleached at room temperature (Table 1). However, optimum enzyme wasextracted when stem pieces were blended and extracted under the sameconditions (see Table 1). Bark and stick of the plant when extracted sepa-rately showed the presence of about four times more amylase activity in stembark than in the stick (see Table 1). Leaves and seed of the plant did notcontain sufficient amount of enzyme (see Table 1). With the above extrac-tion protocol, recovery of amylase in buffer was 470 ± 50 U.g−1 of freshstem. In addition, 3.0 ± 1.5 U of maltase and 1.5 ± 0.5 U of isomaltase.g−1

of fresh stem were extracted simultaneously with amylase. Conversely, fresh

TABLE 1 Distribution of Amylase in Tinospora cordifolia and Enzyme Yield by VariousExtraction Methods

Plant tissue Method of extraction Enzyme recovery

Whole stem a) Leaching for 3 h at(i) 4 ◦C 60 ± 10(ii) 28 ± 2 ◦C (room temperature) 100 ± 20(ii) 50◦C 250 ± 50

(U.g−1fresh tissue)b) Extraction of blended mass (in 0.1M

acetate buffer, pH 5.0) at(i) Room temperature 110 ± 20 (U.g−1 fresh

tissue)(ii) 50◦C 470 ± 50 (U.g−1fresh

tissue)(iii) Extraction of blended mass at 50◦C

followed by ultrafiltration (PM-10) andlyophilization

28,500 ± 500 (U.g–1 ofdry powder)

(iv) Extraction of blended mass at 50◦Cfollowed by ethanol precipitation(4 vol), centrifugation andlyophilization

90,000–1,10,000 (U.g−1 ofsolid)

c) Lyophilization of fresh stem pieces 1,900 ± 200 (U.g−1 dryweight)

d) Fresh stem pieces dried at 40◦C for 24 hand ground to fine powder

1,300 ± 200 (U.g–1 drypowder)

Bark portion Extraction of blended mass (in buffer)at 50◦C

900 ± 50 (U.g−1freshtissue)

Stick portion Do 250 ± 50 (U.g−1 freshtissue)

Seeds Do 23 ± 2 (U.g−1 freshtissue)

Leaves D0 7 ± 2 (U.g−1fresh tissue)

Note: The data expressed are the average of the five sets of experiments.

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stem pieces, dried by lyophilzation and subsequently ground into fine pow-der, gave a crude enzyme preparation containing 1,900 + 200 U amylase.g−1

of powder. Stem pieces dried at 40◦C and ground into fine powder gavea lower potent preparation of 1,300 ± 200 U amylase.g−1 of powder (seeTable 1). The ethanol precipitate on lyophilization, yielded a solid prepara-tion of activity of 90,000 to 1,10,000 U amylase.g−1 of solid (see Table 1).This preparation was stable over a year at room temperature (see Figure 1).The recovery of amylase activity by ethanol precipitation was satisfactory(> 90 %), while those of maltase and isomaltase were about 50% lower(Table 2).

Catalytic Properties of the Crude Enzyme

Table 2 describes enzyme activities of the crude aqueous extract ofT. cordifolia and that of ethanol-precipitated enzyme preparation. It appearsfrom the table that the crude preparation had substantial amount of maltase,

TABLE 2 Action of Crude Extract of Tinospora cordifolia on Substrates

Specific activity of enzyme ( U.g−1 protein)

SubstratesCrude aqueous enzyme

extractEthanol precipitatedconcentrated enzyme

Maltose (1% w/v) 0.654 0.34Isomaltose (1% w/v) 0.339 0.165P-nitrophenyl-α-D-glucoside

(0.1%w/v)0.480 0.250

1-O-methyl-α-D-glucoside (1% w/v) 0.354 0.172Raffinose (1% w/v) 1.50 0.73Trehalose (1% w/v) 0.096 0.050Melezitose (1% w/v) 0.45 0.27Nigeran (0.5 % w/v) 0.155 0.083Inulin (1% w/v) 0.186 0.089Soluble starch (potato) (1% w/v) 94.2 90.23Amylopectin (1% w/v) 92.6 91.5Glycogen (1% w/v) 47.4 45.5Raw starch (potato) (1% w/v) 9.4 8.4Ungeltainized wheat starch

(1% w/v)7.8 6.9

Ungelatinized corn starch (1% w/v) 4.8 3.9

Note: The results expressed are the average of the triplicate sets of experiments.Amylase activity was determined in 0.1M acetate buffer, pH 5.0 at 50◦C using 1 % (w/v) soluble starch(potato) as substrate. Reducing sugar formed was estimated by dinitro salicylic acid reagent (DNSA).Amylopectin, glycogen and raw starch hydrolysis was carried similarly. Pullulanse, 1-O-methyl-α-D-glucosidase, raffinase, melizitase and inulinase activities were determined in the same way using 1%(w/v) each of pullulan, 1-O-methyl-α-D-glucoside, raffinose, melizitose, and insulin, respectively, assubstrate.Maltase, isomaltase, trehalase and nigeranse activities were determined at pH 5.0 at 37◦C using 1% (w/v)each of maltose, isomaltose, trehalose, and 0.5 % (w/v) nigeran, respectively, as substrates and totalglucose formed was estimated by glucose oxidase–peroxidase (GOD-POD) reagent.

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isomaltase, PNPGase, α-glucosidase, raffinase, trehalase, and melizitase activ-ities. Hoever, recovery of these glucosidases activities was more or less 50%in the ethanol precipitate. Conversely, the plant enzyme was equally activeon soluble starch and amylopectin. Glycogen was also hydrolyzed to theextent of 50% of that of starch by the enzyme. The enzyme also showed lowsaccharifying activity on raw starches and inulin.

Thermo Stability and Acid Stability of CrudeT. cordifolia Amylase

Salivary and pancreatic amylases, although active in a duodenal pH environ-ment, had poor acid stability (Table 3). A. oryzae amylase, commonly usedin pharmaceutical preparation, showed poor acid stability and low activity atpH 3.0 and 7.0. T. cordifolia amylase retained about 35 ± 5% and 72 ± 4%activity at pH 3.0 and 7.0, respectively, compared to its optimal activity atpH 6.0 (see Table 3). The inactivation kinetics at pH 3 (see Figure 2) deter-mined for various amylases showed that T. cordifolia amylase retained morethan 50% activity over a period of 4 hours, whereas other amylases lostactivity in 30 min. T. cordifolia crude enzyme showed optimal activity onstarch in the range of 60◦ to 65◦C, and t1/2 at 62◦C was 30 min, whereasthose for extracellular A. oryzae amylase were 50◦C, and t1/2 at 55◦C was30 min. However, both enzymes are more or less equally active at ambienttemperature.

T.cordifolia Enzyme Supports Glucogenic Digestionof Cereal Starch

T.cordifolia enzyme hydrolyzed various cereal starches in the followingdecreasing order: gram (76%–79%)> corn (72%–75 %)> wheat (60%–65%)> rice (48%–52%) while fungal amylase (A. oryzae) hydrolyzed thesame order of corn (35%–38%)> wheat (28%–32%)> gram (27%–29%)> rice(22%–26%) under similar conditions (Figure 3a and 3b). T.cordifolia enzymemixture released appreciable amount of glucose (more than 50% of reducingsugar) from starch, amylopectin, and glycogen. The plant enzyme signif-icantly hydrolyzed amylopectin and glycogen while fungal enzyme actedpoorly on those substrates (Figure 4a and 4b). The dextrinizing kinet-ics, determined iodometrically, showed that T.cordifolia amylase causeda decrease of 92% to 95%, 88% to 92%, and 84% to 87% in iodinecolor for soluble starch, amylopectin, and crude gelatinized corn starch,respectively, in 40 min while A.oryzae amylase caused a decrease of70% to 73%, 53% to 56%, and 48% to 51% in iodine color for the same(Figure 5).

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TAB

LE3

Cat

alyt

icA

ctiv

ityan

dSt

abili

tyofA

myl

ases

(Var

ious

Sourc

es)

inB

uffer

sat

pH

3to

pH

7

Mea

nen

zym

eac

tivity

%O

ptim

alac

tivity

%Res

idual

stab

ility

(1h)

pH

3pH

4pH

5pH

6pH

7pH

3pH

4pH

5pH

6pH

7

Pla

ntam

ylas

e35

±5

64±

289

±3

100

72±

472

±2

90±

210

094

±3

90±

3Fu

nga

lam

ylas

e(A

.ory

zae)

5±

0.3

45±

310

080

±3

20±

122

±2

47±

287

±2

100

95±

2Pan

crea

ticam

ylas

e0

4±

218

±2

60±

210

05

±1

30±

245

±3

53±

410

0Sa

livar

yam

ylas

e1

±0.

23

±0.

512

±1

66±

210

07

±2

31±

446

±2

59±

310

0

67

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0

20

40

60

80

100

0 30 60 90 120 150 180 210 240Time (min)

% A

ctiv

ity

FIGURE 2 Acid stability (at pH 3.0) Plant amylase, Fungal (A. oryzae) amylase, salivaryamylase, and pancreatic amylase.

Nontoxicity of Crude Enzyme Protein

The crude lyophilized enzyme preparation (28,500 ± 500 U.g−1) was non-toxic at up to a dose of 1.5 g.Kg−1 body weight (about 42,750 U.kg−1 bodyweight). No mortality was observed in the dose range of 0.2 to 1.5 g.Kg−1

body weight. Subacute toxicity was determined by administering orally theabove enzyme preparation in doses of 0.1 to 0.3 g.kg−1 body weight twicedaily for 20 days. However, no mortality was recorded until the forty-fifthday of the study. No behavioral abnormalities were observed in the rats.Body weight and food intake were in the normal range.

DISCUSSION

T.cordifolia as a rich source of extracellular saccharifying amylase was firstreported by Sengupta et al. (31). Later, the enzyme was purified and wasidentified to be a 43-KDa thiol protein (23). In the process for the preparationof commercial enzyme grades from the plant, enzyme extraction protocolfrom plant stem was optimized. The extraction buffer selected was 0.1Macetate buffer, pH 5.0 containing 10 mM CaCl2 as activator of amylase activ-ity, 5 mM EDTA and 10 mM cysteine as protectors of enzyme activity againstheavy metals poisoning and areal oxidation, respectively. Commercial plantenzymes were mostly found to be thiol enzyme–sensitive toward oxida-tion and heavy metal inhibition (15). Since the plant (T.cordifolia) containsgum, extraction of enzyme from stem was low at ambient temperaturebut gradually became high with the increase in temperature upto 50◦C.T.cordifoila amylase was found to be an extracellular one with the bulkactivity (900 ± 50 U.g−1 fresh tissue) present in the bark (see Table 1).However, since manual debarking of stem was a very tedious process forlarge amounts of raw material, entire stem was used for extraction instead ofthe richer source (the bark). Though the storage stability of enzyme in fresh-cut T. cordifolia stem as raw material was not very good, plant stem pieces

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FIGURE 3 Hydrolysis of cereal starch fungal (A. oryzae) amylase and plant (T. cordifolia)enzyme. Plant enzyme on Cornstarch ( ), wheat starch ( ),Gram starch ( ), and Rice starch (

):Fungal enzyme on Cornstarch ( ),wheat starch ( ),Gram starch ( ), and Rice starch ( ).

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FIGURE 4 Amylopectin ( ) and Glycogen ( ) hydrolysis by plant (T. cordifolia) amylase:amylopectin ( ) and Glycogen ( ) hydrolysis by fungal (A. oryzae) amylase.

dried by lyophilization and kept at room temperature in sealed containersretained more or less full enzyme activity over 18 months of storage (seeFigure 1). Various commercial preparations of T. cordifolia enzyme (seeTable 1) showed that the potency of these preparations was better thanthat of malt powder (in terms of enzyme activity), reported to contain 953 Uamylase.g−1 (11). The recovery of maltase and isomaltase was proportionatewith amylase activity as present in crude extract. The recovery of amylaseactivity by ethanol precipitation was satisfactory (>90%), while those ofmaltase and isomaltase were about 50% lower see Table 2). Chilled acetone

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0

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FIGURE 5 Iodometric estimation of dextrinizing activity of T.cordifolia amylase on Solublestarch ( ), Amylopectin ( ), and crude gelatinized corn starch ( ), and by A.oryzae amylaseon soluble starch ( ), amylopectin ( ), and crude gelatinized corn starch ( ).

precipitation did not improve recovery of sucrase, maltase, or isomaltase(data not given). Decrease of substrate specificities of all the disaccharidasesby ethanol precipitation is certainly significant (about 50%). Since recoveryof protein by ethanol precipitation was more or less above 95%, it seems thatdisaccharidases are less stable than amylase when exposed to organic solventfor the period required to collect enzyme by centrifugation at cold. Changeof specific activities of amylase, amylopectinase, or glycogenase may not beconsidered significant, as the decrease percentage average value of the trip-licate sets of experiment is between 1% and 4% (see Table 2). Poor recoveryof amylase by ammonium sulphate precipitation of the crude extract wasreported earlier (31)

Crude A.oryzae amylase did not show any maltase activities as such(data not shown). Production of amylolytic enzymes from microbial sourcesrequires maintained upstream process technology, which is cost-effective,but no such upstream technology is required for extracting plant enzymes.The plant source itself acts like a bioreactor producing the enzyme, whichneeds only to be harvested and extracted. Plant parts such as stem, produc-ing a huge amount of extracellular enzymes, are a cheap source, as theserenewable parts can be harvested time to time. It is important to mentionthat the crude extract was a good source not only of amylase but of impor-tant disaccharidases (maltase, isomaltase, trehalase) not available at low cost.

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The crude extract was also capable of hydrolyzing trisaccharidases suchas raffinose and melezitose and could even hydrolyze ungelatinized rawstarches (see Table 2). Cereals such as rice and corn, which have consider-able amylase activities, are also known to contain α-glucosidase activities(22,27). It is interesting to note that mammalian digestive system con-tains sucrase-isomaltase (EC 3.2.1.48 and 3.2.1.10) and maltase-glucoamylase(3.2.1.20 and 3.2.1.3) complex in the small intestine brush-border membrane(25,28), which ensure complete breakdown of dietary starch into glucose,which can then be absorbed into the blood stream.

Acid stability is a highly desired property of an amylase to be used asa digestive enzyme. An ideal digestive amylase should be stable and activeover a wide pH range varied during starch digestion from stomach to duode-num (13,39). T. cordifolia amylase showed better acid stability than fungal,pancreatic, and salivary amylases (see Table 3 and Figure 2). It retainedalmost more than 50% of its initial activity even after 4 h of incubationat pH 3 while fungal amylase almost completely lost its catalytic activity.Thermal stability of commercial enzyme is essential for low-cost down-steam processing and storage. T.cordifolia amylase displayed better thermalstability than the most commonly used digestive amylase (A. oryzae) .

Edible cereals and tubers contain various amounts of amylopectin instarch (e.g. 74% –76%, 75%, 74%–78%, 70% –80%, 70%–80%, 80%, 99%in corn, wheat, barley, sorghum, potato, medium grained rice, and waxycorn, respectively (8,20). Consequently, cereals are not equally degradedby α-amylases, which stops acting before reaching α-1,6 branch point ofamylopectin. It may be mentioned that starch containing both amylose andamylopectin is digested in two steps invoving dextrinization of starch (byhydrolysing only α-1,4 linkages) and saccharification of dextrins into glu-cose (hydrolysing both α1,4 and 1,6 linkages). In the commercial processfor saccharification of cereal starches into glucose, numbers of starch-degrading enzymes are used as per manufacturer’s protocols. Enzymes suchas dextrinizing and saccharifying amylases, amyloglucosidase, pullulanase,and debranching enzymes are used in various combination to hydrolyseboth α-1,4 and 1,6 linkages and obtain glucose (26). T. cordifolia amylaseconversely could hydrolyze both starch and amylopectin with the same easeand could even digest glycogen much better than fungal enzyme (Figure 3aand 3b; Figure 4a and 4b). It has been reported that pea leaves and potatotuber amylases also showed ability to hydrolyze both soluble starch andamylopectin equally (41,42).

The crude T. cordifolia enzyme showed twofold more saccharifyingactivity on various cereal starches (corn, wheat, gram, and rice) compared tothat of the same amount for fungal enzyme. It produced more reducing sugarand glucose from gelatinized cereal starches. Human glucogenic digestion ofstarch require a consortium of enzymes; salivary and pancreatic amylases(α-1.4 hydrolyzing) for dextrinization and intestinal brush border membrane

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protein sucrase-isomaltase (SI) and maltase-glucoamylase complex (MGAM)for conversion of dextrins into glucose entering into blood stream (25,28).Isomaltase accounts for approximately half the digestion of maltotriose andmaltose, both of which are products of amylolytic digestion of dietary starch(17). The presences of disaccharidases along with amylase assist in com-plete hydrolysis of starch into glucose. A.oryzae amylase actually substitutedextrinizing enzyme of human digestive system but depends on the activityof brush border enzymes for putting glucose into blood stream. It has alreadybeen mentioned that T.cordifolia extract contains appreciable amount ofmaltase, isomaltase, and other disaccharidase activity, thus mimicking theenzyme consortium of mammalian digestive system. Therefore, its traditionaluse to treat indigestion (as mentioned in Ayurveda) has a strong scientificsupport. The crude enzyme did not cause formation of any limit dextrinas evident by full decolorization of iodine color and presence of glucose,maltose, and lower oligosaccharides detected on a thin-layer chromatogra-phy plate (data not shown). Thus, it may be suggested that this enzymepreparation is a complete substitute of human starch digestive enzymes.T.cordifolia enzyme, unlike fungal enzyme, may be beneficial for personswith sucrase-isomaltase defficiency, suffering from osmotic diarrhea (4,40).

T. cordifolia has been used for a long time in Ayurvedic medicine,and several toxicity tests (10,18) have proved it practically nontoxic evenat a high dose of 3-gm/kg body wt. (1). The present study, undertaken todetect the acute toxicity level of the enzyme protein, showed that the crudelyophilized enzyme preparation (28,500 ± 500 U.g−1) was nontoxic at upto a dose of 1.5 g .Kg−1 body weight (about 42,750 U.kg−1 body weight).The dosage amount is quite high as far as a digestive enzyme preparationis concerned. The presence of allergen in fungal spore possibly remainedin enzyme preparations as contaminant, and its use over a long period in abakery has reported to cause allergic symptoms to the workers (36). Thus,T.cordifolia may be a potential commercial source of plant digestive enzymehaving merits over the current commercial fungal digestive amylase.

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Documents

Evaluation of Tinospora cordifolia Amylase as a Commercial Digestive Enzyme of Plant Origin