Journal of the Science of Food and Agriculture J Sci Food Agric 80:1343±1350 (online: 2000)

Production, partial purification and propertiesof b-mannanases obtained by solid substratefermentation of spent soluble coffee wastesand copra paste using Aspergillus oryzae andAspergillus niger†

Carlos Regalado,1* Blanca E Garcıa-Almendarez,1 Luz M Venegas-Barrera,1

Alejandro Tellez-Jurado,2 Gabriela Rodrıguez-Serrano,2 Sergio Huerta-Ochoa2 andJohn R Whitaker31DIPA-PROPAC, Facultad de Quımica, Universidad Autonoma de Queretaro, CU Cerro de las Campanas S/N, Queretaro, 76010 Qro,Mexico2Depto Biotecnologıa, Universidad Autonoma Metropolitana-Iztapalapa, Av Michoacan y la Purısima S/N, Col Vicentina, Deleg Iztapalapa,CP 09340, Mexico3Department of Food Science and Technology, University of California, Davis, CA 95616, USA

(Rec

* CoCam† PapContCont

# 2

Abstract: In order to achieve a higher added value of two galactomannan-containing wastes, copra

paste and spent coffee from the soluble coffee industry (SCW), solid substrate fermentation (SSF) was

used. Filamentous fungi Aspergillus oryzae and A niger were used to evaluate the feasibility of

producing b-mannanase by SSF. A 23 factorial design was used to select the best interaction among the

two fungi, the two substrates and two fermentation times. The treatment `A niger±copra±2.5 days'

produced a signi®cantly higher (p<0.05) b-mannanase activity, having ®ve different isoforms of the

enzyme, one of which was partially puri®ed to a speci®c activity of 764Umgÿ1 (U=nmol of mannose

released per second from a galactomannan substrate). Copra paste had a higher mannose/galactose

ratio (14:1) than SCW (6:1), and low oil content, which led to higher b-mannanase production from

SSF. A b-mannanase from SSF of copra produced by A oryzae was highly puri®ed using acetone

precipitation and cation exchange and size exclusion chromatographies. This enzyme had an MW of

110kDa, a pI between 3.5 and 4.5 and a speci®c activity of 1760Umgÿ1; puri®cation achieved was 90.7

times. The temperature and pH for optimal activity were 40°C and 6.0 respectively. The optimal

temperature was lower and the optimal pH higher than others previously reported (produced by

submerged fermentation), which could be important for viscosity reduction of concentrated coffee

extract in instant coffee manufacture. Copra is an interesting alternative for b-mannanase production,

since it is readily available in Mexico; moreover, the residue after SSF has a reduced galactomannan

content and may be used for monogastric animal feed.

# 2000 Society of Chemical Industry

Keywords: solid substrate fermentation; Aspergillus niger; Asperzillus oryzae; copra paste utilisation; coffee wastesutilisation; b-mannanase

INTRODUCTIONIn 1995, Mexico produced about 600tons of coffee

wastes from the soluble coffee industry and about

33000 tons of copra paste (waste of the coconut oil

industry).1 Both wastes are good sources of hemi-

cellulose (b-1,4-hetero-mannan, b-1,4-hetero-xylan

and other substituents in positions 2 or 6 such as

arabinose, galactose, glucuronic acid and acetyl-

mannobiose heteropolymers).

eived 31 August 1998; revised version received 2 February 2000; acc

rrespondence to: Carlos Regalado, DIPA-PROPAC, Facultad de Qpanas S/N, Queretaro, 76010 Qro, Mexicoer presented in part at the IFT meeting in Atlanta, GA, 1998

ract/grant sponsor: CONACYTract/grant sponsor: CONCYTEQ

000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2

Endo-1,4-b-D-mannanases (EC 3.2.1.78) are a kind

of hemicellulases, and most are glycoproteins. They

can randomly hydrolyse the 1,4-b-D-mannosidic

linkages within the main chain of mannans and

heteropolysaccharides consisting mainly of mannose,

such as galactomannans or glucomannans, producing

manno-oligosaccharides. The extent of hydrolysis

depends on the degreee of substitution and the

distribution of the substituents. Hydrolysis of gluco-

epted 18 February 2000)

uımica, Universidad Autonoma de Queretaro, CU Cerro de las

000/$17.50 1343

C Regalado et al

mannans is affected by the proportion of glucose and

mannose units.2,3

Mannanases are used in fruit and vegetable macera-

tion, wine and juice clari®cation, oil extraction from

legume seeds and the reduction of viscosity of coffee

extract in instant coffee manufacture.4,5

Solid substrate fermentation (SSF) is a simple

technique requiring low capital investment and low

energy supply. Water content ranges from 120 to

700g kgÿ1 (depending on the material) and promotes

a relatively low water activity which favours the growth

of ®lamentous fungi inoculated into the wet solid

substrate.6 SSF is a heterogeneous system involving

three phases: solid, liquid and gas. Factors affecting

SSF are gas diffusion, heat transfer, material porosity,

particle size and substrate consistency, leading to poor

control of pH and temperature.7,8

Copra and SCW can be used as support materials

and carbon sources in SSF, and b-mannanase can be

produced using the ®lamentous fungi A oryzae and Aniger. These fungi do not produce toxins, their

metabolic products enjoy GRAS status, and they can

be used in the food industry.9 The solid matter from

SSF may be used as an alternative probiotic ingredient

in cattle feed because of the single cell protein and high

®bre content.

The objective of this study was to give added value

to copra and coffee wastes by incorporating them into

a solid state fermentation process using A oryzae and Aniger to produce b-mannanases. Experiments were

conducted to select the most appropriate combination

of fungus, substrate and fermentation time leading to

the highest b-mannanase production. Partial puri®ca-

tion and characterisation of the produced b-manna-

nases are also reported here.

MATERIALS AND METHODSMaterialsAll chemicals, including analytical-grade salts, were

purchased from Sigma (St Louis, MO, USA), except

those used for electrophoresis which were supplied by

Bio-Rad (Hercules, CA, USA). Deionised water

obtained from Milli-Q equipment (Millipore, Bed-

ford, MA, USA) was used in all experiments. Coffee

wastes were supplied by Nestle (Toluca, Mexico), and

copra paste by Hidrogenadora Nacional (Tlalnepan-

tla, Mexico).

Aspergillus oryzae CECT2094 was supplied from the

Spanish-type culture collection (Universidad de

Valencia, Spain), while Aspergillus niger UAM-GS1

was isolated from copra paste and supplied from the

culture collection of the Metropolitan University

(UAM-I, Mexico).

MethodsStrain identi®cation was carried out using the micro-

culture technique as reported by Samson and Pitt.10

Cotton blue stain was used to monitor the shape and

growth of both fungi. They were incubated on slants of

1344

potato dextrose agar for 4 days at 30°C, followed by

spore harvesting using 1glÿ1 Tween 80. Microscopic

count was accomplished using a Neubauer chamber

(Bright line Hausser).

Crude protein, moisture content, ether extract and

chlorogenic acid were assayed according to the

methods of the AOAC.11 Soluble protein was quanti-

®ed using the method of Bradford12 with bovine serum

albumin as standard.

Mannanase activity was determined using carob

bean ¯our (from Ceratonia siliqua) as substrate

(5glÿ1), dissolved in 50mM acetate buffer, pH 5,

stirred overnight, centrifuged for 10min at 15000�g,

and the supernatant was stored in the cold (4°C). The

enzyme was incubated with this supernatant at

50�0.5°C for 10min and the reaction was stopped

by immersing the samples in iced water kept at 0±4°C.

The incubation temperature was determined from

preliminary tests conducted between 50 and 80°C,

according to a survey on the reported optima.13±15 The

reducing sugars produced by the b-mannanases were

quanti®ed using the method reported by Sinner and

Puls,16 from a mannose standard curve (1±10mM). To

improve precision and minimise risk of interferences,

calibration blanks were used for substrate, broth and

buffer solutions. An activity unit (U) was de®ned as

nmol of mannose released per second from the

galactomannan substrate. Proteolytic activity was

measured by the casein digestion method17 and

expressed as ng of tyrosine released per second from

the casein substrate.

Fermentation columnsGlass columns (22�190mm), submerged in a con-

stant-temperature water bath were used as the waste

solids support,18 with a packing density of 0.6gcmÿ3,

55g kgÿ1 moisture content and an inoculum of 1�106

spores gÿ1 dry matter. A water vapour-saturated

stream of air ®tted with rotameters controlled air ¯ow.

A high-air-¯ow condition (20cm3minÿ1), producing

no ¯ooding of the column, was chosen. The waste

solids were preheated at 70°C for 30min to facilitate

fungal penetration. Since there was no mixing, the

temperature and pH inside the columns could not be

controlled over the retention time. Thus they were

initially adjusted to yield high b-mannanase produc-

tion in preliminary runs, and these conditions were

®xed at 30°C and 5.4 respectively.

Experimental designOwing to thorough washing during soluble coffee

extraction, the resulting SCW had to be supplemented

to support microbial growth and thus mannanase

production. The mineral supplementation (glÿ1) was

0.01 FeSO4 �7H2O, 0.05 CaCl2, 7.54 K2HPO4 and

2.32 KH2PO4, while nitrogen addition was as 13glÿ1

yeast extract (Difco, Detroit, MI, USA) and 4glÿ1

ammonium sulphate.19 A 23 factorial design, with

three replicates, was carried out to determine the

highest b-mannanase activity resulting from the inter-

J Sci Food Agric 80:1343±1350 (online: 2000)

Table 1. Proximal analysis of soluble coffee waste and copra paste (averageof three replicates � standard deviation)

Parameter

Soluble coffee waste

(g kgÿ1)

Copra paste

(g kgÿ1)

Ether extract 285�2 55�1

Total solids 962�1 930�1

Protein (N�6.25) 166�2 200�6

Reducing sugars 1�0.3 190�2

Chlorogenic acid 34�1 ND

ND, not determined.

b-Mannanases from solid substrate fermentation

actions between the two substrates (SCW and copra)

and the two fungi (A niger and A oryzae) at two

different fermentation times (2.5 and 5 days). The

resulting data were analysed using the SAS software,

version 6.12, including the Tukey test of signi®cant

difference among treatment means.

Crude extractA 50mM acetate buffer, pH 5.0, was added to the

fermented material in a 1:1 (v/v) ratio and the mixture

was pressed at 13.8MPa (ERKCO press, model PH-

51T, Aeroquip, Toluca, Mexico). The ®ltrate was

centrifuged at 15000�g and 4°C for 15min using a

Beckman J2-MC centrifuge (Beckman Instruments,

Wilmington, DE, USA). A serine-protease inhibitor

(phenylmethyl-sulphonyl ¯uoride, PMSF, 1mM), was

incorporated in the extracting buffer to reduce activity

losses. This inhibitor was also used when dialysis was

required.

PurificationCold acetone precipitation was conducted using a 3:1

volume ratio of acetone/extract. After centrifugation,

conducted under the same conditions as for the crude

extract ®ltrate, the supernatant and the resuspended

precipitate were dialysed in membranes of 12kDa

molecular weight cut-off (MWCO; Sigma) for 48h at

4°C. Activity was determined in both fractions.

Alternatively, the pressed extract was directly concen-

trated in a Minitan (Millipore) tangential ultra®ltra-

tion unit using a 10kDa MWCO membrane at 4°C for

16h. Samples were freeze-dried using Virtis 5L

equipment (Gardiner, NY, USA).

Before injection into a chromatographic column the

samples used were dialysed against the column buffer.

A DEAE-cellulose column was ®tted to a Gradifrac

system (Pharmacia, Uppsala, Sweden) equilibrated

with 20mM Bis-Tris buffer, pH 6.0. A 2ml portion of

extract was injected and 4ml fractions were collected

at a ¯ow rate of 1mlminÿ1; elution was conducted

using the same buffer with a linear gradient of 0.5M

NaCl from 72 to 128min (0%±50%), kept constant at

50% from 128 to 160min, followed by a linear

gradient from 160 to 240min (50±100%). Protein

and enzymatic activity were determined for each

fraction, and those having activity were dialysed

against a 20mM acetate buffer, pH 5.0, and freeze-

dried for further testing.

SP-Trisacryl M (sulphopropyl-poly[N-tris(hydroxy-

methyl) methyl acrylamide]; Sigma) was used for

cation exchange chromatography in a Bio-Rad column

®tted to the Gradifrac system. A 20mM MES buffer

was used to pre-equilibrate the column at pH 6.0.

Elution was performed using the same buffer contain-

ing 1M NaCl with a linear gradient from 40 to 80min

(0±25%), kept constant at 25% from 80 to 100min,

followed by a linear gradient from 100 to 140min (25±

100%). Injection volume, ¯ow rate and fraction size

were the same as before. The pooled activity fractions

were dialysed against 20mM acetate buffer, pH 5.0, to

J Sci Food Agric 80:1343±1350 (online: 2000)

remove the salt, and then freeze-dried. Alternatively, a

Mono Q (Pharmacia) column ®tted to a Biologic LP

(Bio-Rad) system was pre-equilibrated with 20mM

phosphate buffer, pH 7.6. Gradient elution with the

same buffer containing 1M NaCl was used, as

described above, to separate the b-mannanases.

To achieve further puri®cation, a 2.5cm�50cm

column loaded with Sephadex G-100 and ®tted to the

Gradifrac system was used with an injection of a 2ml

sample, eluted with 20mM MES buffer, pH 6.0, at a

¯ow rate of 0.5mlminÿ1, and 4ml fractions were

collected.

Gels of 100glÿ1 polyacrylamide were used for native

electrophoresis, while 110glÿ1 polyacrylamide gels

were used for SDS-PAGE.20 Low-molecular-weight

protein markers (10±70kDa; Sigma) were used for

SDS-PAGE and wide-range protein markers (14±

500kDa; Sigma) for native electrophoresis. Coomassie

Blue dye was used to stain the protein bands.

A zymogram was obtained following the method of

BeÂguin.21 Here the protein bands separated after

native electrophoresis were transferred by diffusion

to an agar gel containing substrate and were then

stained with Congo Red. Mannanase activity was

detected as a colourless band in a blue background

obtained by immersing the gel in an acetic acid

solution. A mini-IEF cell (Bio Rad) employing

polyacrylamide gels with ampholites between pH 3

and 10 was used. Samples (2ml) were injected and the

protein bands were stained with 5glÿ1 Coomassie

Blue dye. Wide-range Bio-Rad pI markers (3±10) were

used.

The pH of optimal activity was determined keeping

a constant ionic strength of 0.43M, adjusted with

NaCl, using the following 0.1M buffers: citrate for pH

3.0±6.0, phosphate pH 7.0, Tris pH 8.0 and borate

pH 9.0. The temperature for optimal activity was

investigated between 5 and 80°C using a Haake

circulating water bath (Model K20, Karlsruhe, Ger-

many).

RESULTS AND DISCUSSIONBoth wastes had high protein contents (see Table 1),

and proteases were therefore expected in the crude

extract. Both fungi can be induced to produce b-

mannanases, but other metabolites constitutively

produced are amylases, proteases and lipases by A

1345

Table 2. Analysis of interactions using the Tukey test to determine significantdifferences (p<0.05) and the best b-mannanase-producing treatment(average of three replicates � standard deviation)

Tukey's group Average activity (Umgÿ1) Treatment a

A 113�40 5

B A 83.3�18 1

B A 78.7�8.3 2

B C 62.0�6.4 3

B C 42.6�9.2 6

B C 37.0�5.5 7

C 21.3�0.9 4

C 20.4�1.8 8

a Treatment 1, A oryzae±copra±2.5 days; 2, A oryzae±copra±5 days; 3, A

oryzae±SCW±2.5 days; 4, A oryzae±SCW±5 days; 5, A niger±copra±2.5

days; 6, A niger±copra±5 days; 7, A niger±SCW±2.5 days; 8, A niger±SCW±5

days.

Figure 1. Anion exchange chromatography (DEAE-cellulose) for the crudeextract resulting from SSF of SCW–A oryzae.

C Regalado et al

oryzae,22 while citric acid and pectinases are produced

by A niger.23 Copra waste was apparently a better

substrate because of its high reducing sugar content

(Table 1). On the other hand, copra had a galactose/

mannose ratio of 1:14,15 while that for coffee waste

was 1:6.24 This is important for mannanase induction,

since the lower the branching of this carbohydrate

heteropolymer, the easier is its degradation by this

enzyme. Chlorogenic acid, a kind of tannin, and the

high oil content found in the SCW (Table 1) could

have affected microbial growth. On the other hand,

copra is a porous material having more than twice the

water retention capacity of SCW. Since in SSF there

should not be free liquid, copra might maintain a

higher moisture content providing better conditions

for fungal growth.

Table 2 shows that the interaction of fungus±

substrate±time was important to produce a high

(p<0.05) b-mannanase activity. Tukey's test showed

that the mean b-mannanase activity produced by the

treatment A niger±copra±2.5 days was signi®cantly

higher than the others (Table 2). It was decided to

use the best two treatments to produce and partially

characterise the resulting b-mannanases. However, a

closer look at one of the SCW fermentations was also

considered important to try to make use of this

waste.

The hemicellulose composition of the SCW, to-

gether with the slight increase in pH observed after

SSF (0.5 pH units) and the 100glÿ1 lower ®nal

moisture content (data not shown), was probably

producing stress in both fungi. This might have

favoured the production of constitutive metabolites

(proteases and lipases given the SCW composition)

and limited the production of induced metabolites

such as b-mannanases. Partial puri®cation of the

extract from SCW±A oryzae±2.5 days, obtained by

direct injection of the ®ltered extract into an anion

exchange column (DEAE-cellulose), resulted in the

separation of b-mannanase activity only in the second,

non-retained, peak (Fig 1). The activity yield (% of

initial activity) was low (26%), with a puri®cation

1346

factor of only 2. However, four different fractions

showed proteolytic activity, and therefore this material

appears more promising for protease production and

further studies are needed for their characterisation.

Fermentation of A niger–copra–2.5 daysAbout half of the initial activity was lost when this

extract was concentrated by ultra®ltration, probably

because of poor enzyme stability and the presence of

proteases other than those inhibited by PMSF. A

sample was passed through a desalting column (Bio-

Rad P6) where two fractions eluting at the end showed

little activity and were discarded. The pooled sample

was injected into the Biologic LP system ®tted with a

Mono Q column. Five fractions showed b-mannanase

activity and two of them, fractions 4 and 5 which

eluted with the salt gradient, represented 73% of the

total activity (data not shown). These multiple b-

mannanase isoforms are probably synthesised to

degrade the complex hemicellulosic material of the

substrate (copra). Apparently the fungus grows well at

the beginning using the high concentration of reducing

sugars, and later on, when the available sugars are

depleted, b-mannanases are produced, secreted and

start to degrade the hemicellulosic material. This

behaviour has been reported for the basidiomycete

Sclerotium rolfsii in submerged culture.25 Fraction 4

had a puri®cation factor of 15, speci®c activity of

764Umgÿ1 and 550g kgÿ1 protein recovery. Optimal

pH and temperature were 3.0 and 50°C respectively;

preliminary results showed that the molecular weight

is about 56kDa and isoelectric point about 4.9. Work

is in progress to further purify this fraction and con®rm

these properties.

Fermentation of A oryzae–copra–2.5 daysCold acetone was used to precipitate the protein in the

crude extract. The precipitate was resuspended in

50mM acetate buffer, pH 5.0, and dialysed against the

same buffer using PMSF as protease inhibitor. A

zymogram (Fig 2, lanes 3 and 4) shows that the crude

extract had several isoenzymes, while the precipitated

J Sci Food Agric 80:1343±1350 (online: 2000)

Figure 2. Native electrophoresis of copra–A oryzae SSF: lane 1, chickenegg albumin; lane 2, bovine serum albumin; lanes 3 and 4, zymogram ofcrude extract and acetone-precipitated extract respectively; lane 5,acetone-precipitated extract; lane 6, purified b-mannanase.

Figure 3. Cation exchange chromatography (SP-Trisacryl M) for theextract from SSF of copra–A oryzae. Buffer MES pH 6.0, 20mM, 1mlminÿ1.

Figure 4. Size exclusion chromatography of the partially purifiedb-mannanase (copra–A oryzae). Buffer MES pH 6.0, 20mM, 1mlminÿ1.

b-Mannanases from solid substrate fermentation

extract had only three activity bands with molecular

weights of about 200, 185 and 110kDa (obtained from

a calibration curve with molecular weight standards).

Preliminary puri®cations were conducted using

anion and cation exchange chromatography at differ-

ent pHs. The best separation was achieved using a

cation exchange column (SP-Trisacryl M) equili-

brated with 20mM MES buffer, pH 6.0. Two peaks

showed b-mannanase activity (Fig 3); one eluted with

Table 3. Purification yields of a b-mannanase produced by SSF of copra by A oryz

Puri®cation step Activity (U) Protein (mg) Speci®c a

Crude extract 483�9.2 24.8�0.38 19

Acetone precipitation 130�3.7 5.30�0.51 24

Cation exchange fraction A1 22.2�1.8 0.45�0.02 10

Cation exchange fraction A2 103�2.8 0.50�0.03 40

Gel ®ltration 49.1a 0.03a 176

a Only one run was conducted using a concentrated sample.

J Sci Food Agric 80:1343±1350 (online: 2000)

the buffer (fraction A1), while the peak eluted with the

salt gradient (fraction A2) had 78% of the recovered

activity.

Since fraction A2 contained most b-mannanase

activity, it was chosen for further puri®cation and

was loaded onto a Sephadex G-100 gel ®ltration

column after dialysis. b-Mannanase activity was found

in a shoulder representing 60mggÿ1 protein, eluted

before a peak without activity, accounting for

940mggÿ1 of the total protein (Fig 4). A puri®cation

factor of 90.7 was achieved; the results of puri®cation

yields up to this step are summarised in Table 3.

Native electrophoresis of this puri®ed fraction showed

three protein bands with molecular weights <132kDa

(Fig 2, lanes 5 and 6). From the zymogram and native

electrophoresis (Fig 2) the band having b-mannanase

activity was identi®ed as that at 110kDa. Since only

one activity band was found in the puri®ed extract, it is

concluded that the other two activity bands shown in

Fig 2 (lane 4) were in fraction A1 (Fig 3).

The molecular weights of b-mannanase obtained

here are higher than those characteristic of other fungi,

including the genus Aspergillus, which range from 30 to

89kDa, Thielavia terrestris NRRL 8126,25Trichodermaharzianum E58,26Trichoderma reesei 27 and the pre-

viously highest, 98kDa, from Aspergillus awamori.28

SDS-PAGE of the puri®ed fraction gave several

protein bands ranging from 26.5 to 36kDa in mol-

ecular weight (results not shown), while the only b-

ae (average of three replicates � standard deviation)

ctivity (Umgÿ1) Puri®cation factor Yield (% of initial activity)

.4�0.90 1.00 100

.1�3.7 1.24 26.8

2�13 5.26 4.60

8�61 21.0 21.2

0 90.7 10.2

1347

Figure 5. pH of optimal activity of the partially purified b-mannanase.

Figure 6. Temperature of optimal activity of the partially purifiedb-mannanase.

C Regalado et al

mannanase in this sample had a molecular weight of

110kDa (see Fig 2), suggesting the presence of

subunits. Kinetic studies on two b-mannanases from

T reesei showed that four subsites are required for their

action on mannooligosaccharides.29

IEF of the puri®ed fraction showed the same three

bands as in Fig 2 (lane 6), in the range 3.5±4.5 (data

not shown), but because of the system used, a

zymogram could not be carried out without destroying

the gel.

The activity of the puri®ed b-mannanase was

strongly in¯uenced by the pH (Fig 5). When the pH

decreased by half a unit from the optimum (pH 6.0),

the activity on both sides was reduced to 50%. From

Fig 5 the activity reported here using pH 5.0 was

underestimated by 60%, and this applies to Figs 3 and

4. Activity was measured at pH 5 because of the high

enzyme stability, and previous reports for other

mannanases indicated that this value was close to the

optimal.13,27 Reported optimal pHs for mannanases

produced by fungi range from 3.2 for one puri®ed

from Sclerotium rolfsii,30 one having two optima (3.0

and 3.8) puri®ed from Aspergillus sp,13 to 5.4 for one

produced by Trichoderma reesei.27 The optimal pH

found here is therefore higher than other reported

optima and allows high b-mannanase activity at near-

neutral pH values.

The temperature for optimal activity was 40°C, but

even at 5°C, 10% of the optimal activity remained,

while no activity was found when the assay was

conducted at 70°C (Fig 6). Optimal temperatures

reported for mannanases secreted by fungi range from

57°C for Streptomyces sp14 to 75°C for mannanases

M3 and M4 from Thielavia terrestris.31 Other optima

are in between, such as 72 and 74°C for two

mannanases of Sclerotium rolfsii when glucomannan

was used as inducer,30 and one from Aspergillus sp

(65°C).13 The optimal temperature of a b-mannanase

from Aspergillus tamarii was not fully evaluated, but its

maximum stability was at 37°C.32 Thus our puri®ed

b-mannanase has one of the lowest reported optimal

1348

temperatures and could be used ef®ciently in processes

where relatively low temperatures are required, such as

viscosity reduction of concentrated liquid coffee

extract to reduce energy costs in instant coffee manu-

facture.33 This enzyme could also be used to improve

the nutritional value of soybean meal or copra paste for

monogastric animals, which has been attributed to a

partial hydrolysis of the b-mannan content of the

feed.34

The activity of puri®ed b-mannanase followed

Michaelis±Menten kinetics, and Km and Vmax were

calculated using the equation of Lineweaver±Burk.

Km=102mM (assuming a locust bean gum molecular

weight of 310kDa) and Vmax=37300Umgÿ1, with a

speci®city coef®cient of 366Umgÿ1mMÿ1, expressed

as Vmax/Km. The Km value is high and the speci®city

coef®cient low compared to puri®ed mannanases

obtained by submerged fermentation using Entero-coccus casseli¯avus and Trichoderma reesei,35,36 but

similar to that produced by submerged fermentation

of Polyporus versicolor.37 No reports were found to

compare these properties with b-mannanase produced

by SSF.

When our highly puri®ed enzyme was tested with a

glucomannan as substrate (konjac gum) the activity

was higher, suggesting that the galactose branches

decrease the catalytic power of this acidic b-manna-

nase. In addition, it might possess a cellulose-binding

domain as has been suggested for a Trichoderma reeseib-mannanase.38

Besides the high b-mannanase activity produced,

the residual solids after SSF may be used for animal

feed. The mycelial protein may have increased the

total protein content of this residue, while the galacto-

mannan has been partially hydrolysed and therefore

may be more digestible by monogastric animals.

Studies on the application of the puri®ed enzyme, as

well as the crude extract having ®ve isoforms, to

J Sci Food Agric 80:1343±1350 (online: 2000)

b-Mannanases from solid substrate fermentation

reduce the viscosity of concentrated coffee extract are

presently being carried out. The multiple isoforms

might work synergistically for this application.

CONCLUSIONSUsing a 23 factorial design, with two ®lamentous fungi

(A niger and A oryzae), two substrates (copra and

SCW) and two fermentation times (2.5 and 5 days), a

relatively high production of b-mannanase activity was

obtained from the treatment `A niger±copra±2.5 days'.

The extract had ®ve isoforms, one of which was

partially puri®ed to a speci®c activity of 764Umgÿ1,

while the puri®cation factor was 15, with a protein

yield of 550mggÿ1.

A b-mannanase obtained by SSF of copra by Aoryzae was highly puri®ed using acetone precipitation

and cation exchange and size exclusion chromatogra-

phies. This enzyme had a molecular weight of

110kDa, an isoelectric point between 3.5 and 4.5

and a speci®c activity of 1760Umgÿ1, while the

puri®cation factor was 90.7, with 10% activity yield.

This is the ®rst report of b-mannanase production by

SSF.

The temperature and pH of optimal activity of the

highly puri®ed b-mannanase are 40°C and 6.0

respectively. The optimal temperature is lower and

the optimal pH higher than those of b-mannanases

previously reported (produced by submerged fermen-

tation), which could be important in reducing the

viscosity of coffee extracts. Km=102mM for locust bean

as substrate, and V =37300Umgÿ1.

m

ACKNOWLEDGEMENTSThanks are given to CONACYT for ®nancial support

through an MSc grant to LMVB and ATJ. Financial

support from CONCYTEQ to CR and from CON-

ACYT to SHO (project 1787 PB) is appreciated.

Thanks are also given to CONACYT for ®nancing the

visiting professorship of JRW.

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