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UTILIZATION OF APPLE PEELS FOR THE PRODUCTION OF PLANT CELL-WALL DEGRADING ENZYMES BY ASPERGILLUS FUMIGATUS MS16.

Byline: H. Jalis, A. Ahmad, S. A. Khan and M. Sohail

ABSTRACT

Apple (Malusdomesticaborkh) is one of the popular fruit with an annual global production of several million tones. The use of apple in food production industry led to the generation of huge waste materials in the form of peels and pomace. Owing to the high amounts of lignocellulosics present in the wastes, these can be served as substrate for the production of plant cell-wall degrading enzymes (CWDE). The present study describes the potential of an indigenously isolated strain of Aspergillusfumigatusto produce plant CWDE using apple peels under submerged as well solid-state fermentation. It was observed that the strain was able to produce higher activity of pectinase and xylanase under both the types of fermentation at 30o as well as at 40oC. While less titers of filter paperase, endoglucanase, -glucosidase was obtained. However, the production of plant CWDE was influenced when the apple peels were supplemented with carboxymethyl cellulose, xylan, pectin and lactose. Interestingly, a structurally irrelevant substrate, lactose, acted as inducer, particularly for the production of pectinase.

Key words: Apple peels, Aspergillusfumigatus, Pectinase, Xylanase.INTRODUCTION

Plant cell-wall, comprised of lignin, cellulose and hemicelluloses is regarded as a renewable chemical feedstock (Kumar et al. 2008). However, the presence of diverse components renders it stable and its biodegradation requires activity of a number of enzymes. Consequently, the production of plant cell-wall degrading enzymes (CWDE) remains a subject of current research. Amongst plant CWDE much of the studies have been conducted on the production of cellulase, xylanase and pectinase because of the multitude of the applications of these enzymes (Ghoraiet al. 2009). Cellulase (endoglucanase, exoglucanase, -glucosidase, together) degrade the most abundant polymer found in plant cell- wall, cellulose to monomers; whereas, xylanase and pectinase act upon constituents of hemicelluloses, xylan and pectin, respectively. Although a number of bacterial species have been reported to produce plant CWDE but fungal enzymes are commercially employed. Because of hyphal mode of growth, fungi can penetrate crude substrates deeply and can elaborate an array of hydrolytic enzymes. Therefore, crude substrates including fruit and vegetable wastes are commonly used for the production of these enzymes (Ghoraiet al. 2009).Apple (Malusdomesticaborkh.) is one of the popular fruit with an annual global production of around59.21 million tonnes (Mahawaret al. 2012). From this production, more than 80% is consumed as table fruit while rest is utilized for value added products including fruit juice production which lead to the generation of huge waste material in the form of peels and pomace. These wastes are rich in lignocellulosics and can serve as substrate for the production of plant CWDE. Whereas a number of literature reports describe potential utility of apple pomace (Mahawaret al. 2012), prospects of utilizing its peels, particularly for the production of fungal enzymes, have scarcely been reported (Berovic' and Ostrovers'nik 1997; Mahawaret al. 2012). Present study describes the utilization of apple peels (AP) for the production of plant CWDE from Aspergillusfumigatus MS16 strain under submerged and solid-state cultivation.

MATERIALS AND METHODS

A. fumigatusMS16 was retrieved from the culture collection of the department of Microbiology, University of Karachi, Pakistan. The strain was selected because of its ability to produce a number of hydrolytic enzymes on commercially purified substrates (Sohailet al. 2009b). Initially, the strain was cultivated in Mandel's medium (Mandelsand Weber 1969) containing different substrates (Table 1) to deduce the induction of a particular CWDE by the substrates. After removing fungal mass, cell-free culture supernatant (CFCS) was used as crude enzyme preparation. Crude enzyme preparation was analyzed for endoglucanase, - glucosidase, filter paperase, xylanase and pectinase activity by adopting the methods described elsewhere (Miller, 1959, Sohailet al. 2009a, b, 2011). One IU of the enzyme was defined as the micromoles of reducing sugars (glucose, xylose or galacturonic acid) liberated from the suitable substrate in one min under standard assay conditions.For the fermentation experiments, apple peels (AP) were collected from a local fruit juice seller, cut into small pieces, crushed and sun dried. The particle size of the peels was maintained by sieving through 100 mesh. The peels (1% w/v) were supplemented in MSM, for submerged fermentation (Smf). Spore suspension (5 x 106 spores/g of substrate) of A. fumigates MS16 was transferred to the flasks containing MSM supplemented with AP and incubated for 8 days at suitable temperature. CFCS was obtained after centrifugation at 5000 g for 20 min and used as crude enzyme preparation.For solid state fermentation (SSF), 2 grams of the AP were placed in 250 ml Erlenmeyer flasks and autoclaved for 30 min. After cooling to room temperature it was moistened with MSM without carbon source to65% moisture level. Fungal spore suspension was inoculated at 5 x 106 spores/g of substrate and incubated at suitable temperature for 8 days. This was followed by an addition of 50 ml of sterile sodium acetate buffer (50 mM; pH 4.8) containing 0.02% Tween 80 to each flask and agitated at 150 rpm for 2 hours. The slurry was filtered through four layers of muslin cloth and filter paper. The filtrate was centrifuged at 5000 g for 20 min and used as crude enzyme preparation.In all fermentation experiments either, 0.5% w/v of carboxymethyl cellulose (CMC), pectin, xylanor lactose were supplemented separately in mineral salt medium to study the effect of additional carbon source on the production of plant CWDE.

RESULTS AND DISCUSSION

The data suggests thatA. fumigates when grown in###MSM###supplemented###with###commercially###purified carbon sources was able to produce###cellulase (- glucosidase, endoglucanaseand filter-paperase), xylanase and pectinase (Table 1); however, the expression of these enzymes varied###with the###nature of substrates. For instance, significant titers of cellulases were observed when the medium was supplemented with Avicel, Sigmacell or carboxymethyl cellulose (CMC); pectin and xylan induced the production of pectinase and xylanase, respectively. However, a co-expression of xylanase was also observed in cellulose containing medium and induction of cellulase was noted in presence of xylan. Interestingly, lactose, a disaccharide that is not a constituent of plant cell-wall, was found as a better inducer of plant CWDE, particularly, forpectinase and xylanase. A previous report also described the role of lactose as inducer for endoglucanase and A-glucosidase (Seiboth et al. 2005), particularly when apple pomace was supplemented with lactose under SmF using Trichoderma sp. GIM 3. 0010 (Sun et al. 2010). Based on these results, lactose, cellulose, pectin and xylanwere used as inducers in fermentation of AP. The most important factor in fermentation is the choice of the substrate (Pandey et al. 2000). Since, AP has not been widely used as a substrate for the production of fungal enzymes, so the main objective of the study was to explore the utility of this substrate for the production of enzymes under submerged as well solid-state conditions. Results indicate that submerged fermentation (Smf) of AP at 30oC yielded higher titers of - glucosidase, pectinase and xylanase (Table 2) which indicatethat AP contains sufficient nutrients for the growth of fungi and production of industrially important enzymes. Furthermore, since the preparation is rich in xylanase and pectinase activities, it can be supplemented in animal-feed to improve utilization of high-forage diet (Ghoraiet al. 2009, Jayaniet al. 2005). It was also noted that the production of pectinase was enhanced when AP was supplemented with pectin or lactose.The submerged fermentation of AP at 40oC gavemore amounts of -glucosidase compared to at 30oC and the activity of this enzyme significantly increased whenAP was supplemented with CMC. It may be due to the fact that the higher temperature facilitates hydrolysis of biological waste (Veeken and Hamelers, 1999) and hence the higher production of enzyme.An earlier report suggests that the production ofpectinase by A. niger under SSF fermentation of apple pomace is advantageous as it yields concentrated product with less effluent (Berovic' and Ostrovers'nik, 1997). The results of the present study under SSF revealed that AP is a good substrate for the production of pectinase at30o and 40oC (Table 3). The production of pectinase at40oC is of some significance as there are very few reports suggesting that###pectinases show optimum activity at temperatures more than 50oC (Gummadi and Panda2003).###It was noted that thexylanase production was decreased when the fermentation temperature was increased from 30o to 40oC, while the production of - glucosidase remained unaffected.The supplementation of AP with CMC resulted in an up-regulation of -glucosidase, pectinase and xylanase production when the SSF was carried out at30oC. Similar finding was also made by Sun et al. (2010) where supplementation of CMC to apple pomace enhanced###the###production###of###endoglucanase###and###A-glucosidase by Trichodermasp. GIM 3.0010 under SSF at30C. Mamma et al. (2008) reported about the production of more amounts of pectinase compared to xylanase or cellulase while studying the multienzyme preparation of A. nigerduring SSF of orange-peels. Similar finding was made by Botella et al. (2005) in SSF of grape pomace by A. awamori, higher titers of pectinase and xylanase were obtained while cellulase production was inhibited.

Table 1. Production of plant cell wall degrading enzymes in presence of commercially purified substrates

###supplemented to Mineral salt medium.

Substrates###Enzymes (IU/ml)

###-glucosidase###Endoglucanase###Filterpaperase###Pectinase###Xylanases

Glucose###0###0###0.016 0.008###0.0638 0.008###0.051 0.089

Lactose###0.057 0.039###0.028 0.009###0.003 0.006###0.664 0.183###0.142 0.135

Maltose###0.057 0.039###0.007 0.012###0.010 0.011###0.372 0.322###0.109 0.095

Sucrose###0###0.128 0.031###0.010 0.010###0.403 0.032###0.342 0.069

Avicel###0.054 0.047###0.030 0.004###0.025 0.006###0.424 0.071###3.179 1.888

Starch###0###0.074 0.074###0.009 0.007###0.471 0.122###0.149 0.133

Pectin###0.107 0.014###0.039 0.010###0.006 0.002###11.095 0.522###0.114 0.099

Sigma cell###0.023 0.027###0.041 0.020###0.016 0.006###0.827 0.167###1.349 1.599

Carboxy Methyl cellulose###0.043 0.018###0.180 0.006###0###0.680 0.047###0.114 0.066

Cellulose###0###0.013 0.022###0.018 0.016###0.119 0.190###0.141 0.122

Xylan from oat spelt###0.102 0.060###0.096 0.081###0.027 0.016###0.236 0.407###0.266 0.091

Table 2.Production of -glucosidase (BGL), endoglucanase (EG), filterpaperase (FP, pectinase and xylanase by A.

###fumigatusMS16 through submerged fermentation of apple peels (AP) with and without supplementation

###of pectin, lactose, carboxymethyl cellulose (CMC) and xylan.

MSM###Enzyme production

supplemented###30oC###40oC

with###BGL###EG###FP###Pectinase###Xylanase###BGL###EG###FP###Pectinase###Xylanase

AP###0.95###0.05###0.040###1.12###0.62###0.1###0.005###0.05###1.0###0.0050

###0.01###0.003###0.05###0.02###0.055###0###0.03

APCMC###0.050 0.080###0.010###0.6###0.2###0.350.066###0.05###0.0050###0.42###1.5

###0.08###0.07###0###0###0.066###0.18

APPectin###0.050 0.0010 0.0080###5.5###1.25###0.010###0.02###0.005 0###5.0###0.5

###0.47###0.095###0.08###0.14###0.07

APLactose###0.005###0.01###0.040###3.0###0.15###0.170.05###0.005###0.010###2.8###0.43

###0###0.02###0.15###0###00###0.06###0.02

APXylan###0.1###0.07###0.020###0.75###1.75###0.150.08###0.02###0.010###0.65###1.7

###0.02###0.04###0.08###0.21###0###0.3###0.05

Table 3.Production of -glucosidase (BGL), endoglucanase (EG), filterpaperase (FP, pectinase and xylanase by A.

###fumigatusMS16 through SSF of apple peels (AP) with and without supplementation of pectin, lactose,

###carboxymethyl cellulose (CMC) and xylan.

###MSM###Enzyme production at

supplemented###30oC###40oC

###with###BGL###EG###FP###Pectinase###Xylanase###BGL###EG###FP###Pectinase###Xylanase

AP###0.1###0.080###0.0050###0.65###0.1###0.1###0.0050###0.050###1.0###0.0050

###0.05###0.04###0.08###0.01###0.07

APCMC###0.21###0.09###0.010###0.72###0.120###0.040###0.060###0.10###0.38###0.0050

###0.01###0.006###0.1###0.06

APPectin###0.0010###0.0010 0.0010###0.3###0.050###0.0050###0.00500###0.0050###0.0050###0.0050

###0.02

APLactose###0.05###0.080 0.0050###0.8###0.050###0.010###0.01200###0.0120###0.0050###0.025 0

###0###0.06

APXylan###0.15###0.090###0.010###0.51###0.25###0.030###0.050###0.080###0.45###0.31.08

###0.02###0.08###0.01###0.05

Conclusion: Apple peel can be a good source of substrate for the production of cell-wall degrading enzymes.

REFERENCES

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