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Enzyme and Microbial Technology 40 (2007) 452455

Preparation of a crude enzymatic from Bacillus licheniformes E-44 and itsevaluation in the hydrolysis of Saccharomyces cerevisiae cell walls

Manuel P erez a , , Raul Piad a, Grethel Milian a, Mara das Gracas Felipe b, Adrianne Ferreira b,Ismael Maciel de Mancilha b, Marta Laur encio a, Joao Batista de Almeida e Silva b

a Center of Biotechnological Studies, University of Matanzas, Matanzas 44740, Cubab Faculty of Chemical Engineering of Lorena (FAENQUIL), Rodovia-Itajub a-Lorena Km 74.5, Lorena, Sao Paulo, SP, Brazil

Abstract

The microbial growth, endospores production and protease activity was investigated during growth of Bacillus licheniformes E-44 on nutrientbroth medium at 40 C. The total number of bacterial cells (8.8 1010 UFC/ml), endospores production (8 107 UFC/ml) and protease activity(17.8 Anson U/ml) were maximal after 20 h of cultivation. The hydrolytic activity of the produced crude enzymatic preparation was assessedon residual distillerys yeast cream for obtaining a Saccharomyces cerevisiae hydrolysate (SCH). An increase of the concentration of gluco-and manno-oligosaccharides and total reducing sugars was observed during the production of SCH. The highest concentrations of gluco- andmanno-oligosaccharides and total reducing sugars were achieved after 16 and 20 h, respectively. 2006 Published by Elsevier Inc.

Keywords: Bacillus licheniformes ; Hydrolytic enzymes; Saccharomyces cerevisiae ; Gluco-oligosaccharides; Manno-oligosaccharides

1. Introduction

To Bacillus genus belong aerobic microorganisms, Gram-positive and saprophytic that form endospores. They grow wellin simple medium and produced hydrolytic enzymes, such asproteases, mannanases and glucanases, in dependence of theculture medium used [13].

Currently, many economically important industrial enzymesare produced by cultivation of bacteria, such as Bacillus sp.[4,5] . Over 300 tonnes of enzymes, mainly proteases, are beingannually produced from Bacillus sp.Microbialproteasesplay animportant role in biotechnological processes, and they accountfor approximately 59% of the total enzymes used [6].

Bacterial endospores are dormant life forms, which can existin a desiccated and dehydrated state indenitely. The Bacillus

species more suitable forproduction of endospores andenzymesare B. subtilis , B. licheniformes , B. cereus and B. clausii . Those Bacillus species have been used as probiotics in animals andhumans [7,8] . On theotherhand,endosporesformationby Bacil-lus sp.has been extensively studied since they have been used asprobiotic agents for eliminating entropathogenic bacteria in ani-mals and humans [2]. The production of extracellular proteases

Corresponding author. Fax: +53 45 253101. E-mail address: manuel.perez@umcc.cu (M. Perez).

and endospores is associated with the total count of Bacillus . Arelationshipamong thecapacity to form endospores andthe abil-

ity to produce proteases has been found [9,10].S accharomycescerevisiae cell walls are constituted by two layers, an externallayer composed of mannoproteins and an internal one of -1,3-and -1,6-glucan, with small quantities of chitin [11,12] . Forthe hydrolysis of the S. cerevisiae cell wall it is feasible to useenzymatic preparations produced by Bacillus sp. strain B. cir-culans WL-12 was effective for the hydrolysis of -(1,3)- and

-(1,6)-glucans of S. cerevisiae cell wall rendering manno- andgluco-oligosaccharides with prebiotic activity in human healthand animal production [1315] .

The aim of this work was to evaluate the microbial growth,the endospores production and the production of proteolyticenzymes by B. licheniformes E-44 strain and the hydrolyticactivity of the produced enzymatic preparation on S. cere-visiae cell walls for producing a SCH with potential prebioticactivity.

2. Materials and methods

2.1. Strain and growth conditions

Bacillus licheniformis E-44 was grown on nutrient broth medium, whichconsisted of (per liter): 5g of bacteriological peptone, 1g of meat extract, 2 g of yeast extract, 5g of NaCl, pH 7.4.

0141-0229/$ see front matter 2006 Published by Elsevier Inc.

doi:10.1016/j.enzmictec.2006.07.020

mailto:manuel.perez@umcc.cuhttp://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.enzmictec.2006.07.020http://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.enzmictec.2006.07.020mailto:manuel.perez@umcc.cu

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2.2. Determination of total bacteria, endospores production and viable bacteria

Sampling to determine viable cells was prepared by seriated dilution in10 ml of phosphate-buffered saline (PBS). Serial dilutions were then made withPBS, plated on Agar yeast medium plates and incubated at 40 C for 28h. B.licheniformes colonies of strain E-44 were counted. Then, spore suspensionswere heatedto65 C for1 h tokillany residual,non-sporulatedcells. Thenumberof endospores was determining as viable cells.

2.3. Protease production in liquid culture medium

Nutrient broth medium enriched with 1% of casein was inoculated witha 12-h old culture and incubated at 40 C for 28h [16]. After cultivation, theculture broth was ltered and centrifuged at 12,000 rpm for 15 min. The cellfree supernatant was used as enzymatic preparation, where protease activitywas tested.

2.4. Protease activity measurement

Proteaseactivity wasmeasured by themethodof Anson [17]. The absorbanceof the solution was measured at 650 nm and the amounts of tyrosine released inthe reaction were calculated with a standard calibration curve. The enzymaticactivity was expressed in units (U). One U of enzyme was dened as the amountof enzyme that released 1 g of tyrosine from the crude enzymatic preparation.

2.5. Hydrolysis of S. cerevisiae I-34 strain with crude enzyme preparation from B. licheniformis E-44

S. cerevisiae I-34 biomass wasobtained as theresidualyeastcreamaccumu-latedin thebottomof thefermentors in an ethanolproductionplant(Arrechavala,Cardenas, Cuba). Samples of residual yeast cream were hydrolysed using 5%of a crude preparation collected after 12 h of cultivation with B. licheniformesE-44. Thepreparationwas enriched with 95% of alcohol residual cream,pH 6.0.

2.6. Analytical methods

Total reducing sugars were determined by DNS colorimetric method usingglucose as calibration standard [18]. Total carbohydrates were analysed byphenol-sulphuric acid method [19]. Aminic nitrogen was determined poten-tiometrically [20] and total nitrogen by Kjeldal method [21]. The hydrolysisdegree of carbohydrates and proteins was calculated from the relations reduc-ing sugars/total carbohydrates and aminic nitrogen/total nitrogen, respectively[22,23] .

Manno- and gluco-oligosaccharides were separated by liquid chromatogra-phy [24] using sepharose 6B together to concanavaline A (Pharmacia, Uppsala,Sweden). The column was equilibrated using the following tampon: 50 mmTrisHCl, pH 7.4, containing 0.5M NaCl at an elution rate of 0.2 ml/min. Eachfraction of isolated oligosaccharides was subjected to total chemical hydroly-sis with 1ml of TFA to 100 C for 2 h. Mannose and glucose were quantiedby the method of Dubois et al. [25] in order to determine manno- and gluco-oligosaccharides content, respectively.

2.7. Statistical methods

For the statistical treatment of the results, the analysis of variance of simpleclassication and the Duncan [26] test comparison were used.

3. Results and discussion

3.1. Kinetics of B. licheniformis E-44 growth, viablebacteria, sporulation and protease production

The effect of culture medium on total bacteria counting,endospores production and viable bacteria during cultivation

of B. licheniformes E-44 is shown in Fig. 1. Total bacteria

Fig. 1. Dynamicsof thecounting oftotalbacteria( ), endospores ( ) andviablebacteria( ) duringcultivationof B. licheniformis E-44.Dilution: 10 4 .Themath-ematical expression for the microbial populations logarithmic transformationwas: log x + dilution x log10 .

counting increased with the increase of the length of the incu-bation period. The highest value of total cells was achievedafter 20 h of incubation. The highest counting of viable bacteria(2.0 105 FCU/ml) was attained at the eighth hour ( P < 0.05).After that, the number of viable cells began to decrease andreached the minimum (4.0 105 FCU/ml) after 28 h. As canbe seen in the time course of endospores formation in themedium, B. licheniformis E-44 displayed the highest sporula-tion (7.6 107 FCU/ml) ( P < 0.01) at 20 h. After that, a declineof endospores production was observed and a minimal value of 7.1 107 FCU/ml was achieved at the 28th hour.

The proteolytic activity was low during the rst hours of cultivation, but it increased with cultivation time, and reachedthe highest value (17.8U) at the 20th hour ( P < 0.05) ( Fig. 2).However,fromthat timeon, it decreasedconsiderably, especiallyduring the following 4 h. According to these results, proteaseactivity appeared to be dependent upon the growth rate of theculture and endospores production. These results are in goodagreement with previous results obtained for Bacillus sp. strainsby other researchers [27,28] .

Fig. 2. Dynamics of the production of proteolytic enzymes during cultivation

of B. licheniformis E-44. R = 0.98.

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Fig. 3. Dynamics of ( ) aminic nitrogen, ( ) total reducing sugars, ( ) totalcarbohydrates in the solid material and ( ) total protein during the preparationof theSCH hydrolysatewith B. licheniformis E-44crude enzymaticpreparation. R = 0.95.

3.2. Hydrolysis of S. cerevisiae I-34 cell walls with crudeenzymatic preparation from B. licheniformis E-44

The crude enzymatic preparation produced during cultiva-tion of B. licheniformis E-44 was used for producing a yeasthydrolysate, which can be used as prebiotic in animal feed[29,30] . For that aim, residual yeast cream obtained after ethano-lic fermentation in a distillery was incubated with B. licheni- formis E-44 crude enzymatic preparation. As shown in Fig. 3,the crude preparation was effective for hydrolysing proteinsand polysaccharides contained in yeast cells. Hydrolysis startedreadily, as can be deduced from the formation of total reduc-

ing sugars and aminic nitrogen, which was evident since thebeginning of the incubation. The concentration of both aminicnitrogen and total reducing sugars increased steadily during therst 12 h of hydrolysis. After that, aminic nitrogen reached amaximum of 42 mg/ml and then started to decrease slightly,apparently because protein hydrolysis had concluded and someamino acids could be used by B. licheniformes cells. On theother hand, the concentration of total reducing sugars achievedapproximately 16 mg/ml, and become constant during the last12 h of incubation. Content of total nitrogen and total carbohy-drates in the solid yeast biomass decreased through the incu-bation due to their solubilisation as they were hydrolysed bythe hydrolytic enzymes contained in the crude enzymatic prepa-ration. Another indication of the hydrolysis of polysaccharidesis the formation of gluco- and manno-oligosaccharides, whichfollowed approximately the same trend that was observed fortotal reducing sugars ( Fig. 4). However, while the formation of total reducing sugars ceased after 12 h, the formation of gluco-and manno-oligosaccharides continued for four more hoursand reached maximum values of 1.4 and 0.8mg/ml, respec-tively, after 16 h of incubation. R =0.97 (P < 0.05). The factthat the formation of oligosaccharides persisted while forma-tion of reducing sugars had nished indicates that the completehydrolysis of polysaccharides was limited by the last step, e.g.the hydrolysis of gluco- and manno-oligosaccharides to glucose

and mannose, respectively. This result is an indication of that

Fig. 4. Formation of gluco-oligosaccharides ( ) and manno-oligosaccharides( ) during the preparation of the SCH with B. licheniformis E-44 crude enzy-matic preparation. R = 0.97.

the crude enzymatic preparation obtained from B. licheniformis

E-44 has higher activity of endoglucanase and endomannanasethan that of -glucosidase and -mannosidase. It is noteworthy,that the low -glucosidase and -mannosidase activity is not adrawback of thecrude enzymaticpreparation,since it is aimed tobe used for producing oligosaccharides-rich hydrolysates withprebiotic activity.

4. Conclusion

These results revealed that by cultivation of B. licheniformesE-44 it is possible to obtain a crude enzymatic preparation thatdisplays theability of hydrolysing S. cerevisiae cellwallsrender-

ingan oligosaccharides-rich hydrolysate withpotentialprebioticactivity.

Acknowledgments

This work was supported by CAPES-MES program (GrantNo. 10/2004). Technical andscientic support of FAENQUIL isacknowledged.Dr. Ram onBocourt (Instituteof Animal Science,Havana, Cuba), Dr. Carlos Mart n and Dr. Reynaldo Villalonga(University of Matanzas, Cuba) are thanked for their criticalreading of the manuscript.

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