Pokušalo se proizvesti različite pektinolitičke enzime submerznim uzgojem s pomoću aktinomicete Streptomyces lydicus. Određena je aktivnost poligalakturonaze i pektin-liaze u supernatantu, ali je utvrđeno da soj nije uspio proizvesti pektin-esterazu. Istražena je proizvodnja poligalakturonaze submerznim uzgojem, uzgojem u polučvrstoj i na čvrstoj podlozi. Svi su pokusi provedeni u statičnoj kulturi i na tresilici. Uzgoj na statičnoj čvrstoj podlozi dao je najbolje rezultate. Kao čvrsta podloga upotrijebljeni su razni poljoprivredni otpaci, a najbolji rezultati postignuti su s pšeničnim posijama.Production of different pectinolytic enzymes was attempted using the actinomycete strain Streptomyces lydicus in submerged fermentation. Polygalacturonase and pectin lyase activities were detected in the culture supernatant, but the strain was not able to produce pectin esterase. Polygalacturonase production was studied in submerged, slurry-state and solid-state fermentation systems. All the experiments were carried out under static and shaking conditions. Solid-state fermentation under static condition was found to be promising. Various agroindustrial residues were tried as substrates for solid-state fermentation. Wheat bran was proved to be the best substrate

Nicemol Jacob

Parukuttyamma Prema

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FTB 44 (2) 263-267. Utjecaj postupka uzgoja na proizvodnju poligalakturonaze s pomoću  novog soja Streptomyces lydicus  Sažetak  Pokušalo se proizvesti različite pektinolitičke enzime submerznim uzgojem s pomoću aktinomicete Streptomyces lydicus. Određena je aktivnost poligalakturonaze i pektin-liaze u supernatantu, ali je utvrđeno da soj nije uspio proizvesti pektin-esterazu. Istražena je proizvodnja poligalakturonaze submerznim uzgojem, uzgojem u polučvrstoj i na čvrstoj podlozi. Svi su pokusi provedeni u statičnoj kulturi i na tresilici. Uzgoj na statičnoj čvrstoj podlozi dao je najbolje rezultate. Kao čvrsta podloga upotrijebljeni su razni poljoprivredni otpaci, a najbolji rezultati postignuti su s pšeničnim posijama. 

Croatian

Influence of Mode of Fermentation on Production of Polygalacturonase by a Novel Strain of Streptomyces lydicus

Production of different pectinolytic enzymes was attempted using the actinomycete strain Streptomyces lydicus in submerged fermentation. Polygalacturonase and pectin lyase activities were detected in the culture supernatant, but the strain was not able to produce pectin esterase. Polygalacturonase production was studied in submerged, slurry-state and solid-state fermentation systems. All the experiments were carried out under static and shaking conditions. Solid-state fermentation under static condition was found to be promising. Various agroindustrial residues were tried as substrates for solid-state fermentation. Wheat bran was proved to be the best substrate

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ISSN 1330-9862 original scientific paper(FTB-1684)Influence of Mode of Fermentation on Production ofPolygalacturonase by a Novel Strain of Streptomyces lydicusNicemol Jacob and Parukuttyamma Prema*Biotechnology Division, Regional Research Laboratory (CSIR), Trivandrum 695 019, IndiaReceived: November 30, 2005Accepted: March 22, 2006SummaryProduction of different pectinolytic enzymes was attempted using the actinomycetestrain Streptomyces lydicus in submerged fermentation. Polygalacturonase and pectin lyaseactivities were detected in the culture supernatant, but the strain was not able to producepectin esterase. Polygalacturonase production was studied in submerged, slurry-state andsolid-state fermentation systems. All the experiments were carried out under static andshaking conditions. Solid-state fermentation under static condition was found to be promi-sing. Various agroindustrial residues were tried as substrates for solid-state fermentation.Wheat bran was proved to be the best substrate.Key words: Streptomyces lydicus, polygalacturonase, pectin lyase, pectin esterase, submergedfermentation, slurry-state fermentation, solid-state fermentationIntroductionPectic substances, present in the primary cell walland middle lamella of higher plants, contribute to thefirmness and structure of plant tissues (1). Different pec-tinolytic enzymes are involved in the breakdown of pec-tin and are widely distributed in higher plants and mi-croorganisms. They are important for plants as they helpin cell wall extension and fruit softening. They have arole in maintaining ecological balance by causing decom-position of plant materials.According to the reaction mechanisms, pectinasescan be classified as esterases, eliminative depolymerases(lyases) and hydrolytic depolymerases (polygalacturona-ses). Pectin esterase (E.C. 3.1.1.11) catalyses the hydroly-sis of methylated carboxylic ester groups in pectin into pec-tic acid and methanol. Pectin lyases (E.C. 4.2.2.10) cleavea-(1,4)-glycosidic linkages by transelimination, which re-sults in galacturonide with a double bond between C-4and C-5 at the non-reducing end. Polygalacturonases areinvolved in the hydrolysis of a-(1,4)-glycosidic linkagesin homogalacturonans. Endo-polygalacturonases (E.C. 3.2.1.15) bring about random hydrolysis of the polymer,whereas exo-polygalacturonases (E.C. 3.2.1.67) act se-quentially from the non-reducing end.The commercial preparations of pectinases are pro-duced mainly from fungi, especially Aspergillus niger (2).Microbial pectinases account for 25 % of the global foodenzyme sale (3). Applications of pectinases include fruitjuice extraction and clarification, wine processing, oil ex-traction, coffee and tea leaf fermentation, retting and de-gumming of fibers, etc. (4).Pectinases are produced by submerged (SmF) andsolid-state fermentation systems (SSF) (5–8), whereasslurry-state fermentation (SLSF) has rarely been report-ed (9). Studies have been conducted on comparative pro-duction of pectinases in systems of SmF and SSF (10).SSF has several advantages over SmF system such ashigher concentration of products, less effluent genera-tion, requirement for simple equipments, etc. (11). Re-ports are very few on the comparison of SmF, SSF andSLSF for the production of pectinases. The present studyinvolves the detection of different pectinolytic activitiesof a novel strain of Streptomyces lydicus and the compari-263N. JACOB and P. PREMA: Pectinases from Actinomycetes, Food Technol. Biotechnol. 44 (2) 263–267 (2006)*Corresponding author; Phone: ++91 471 25 15 325; Fax: ++91 471 24 91 712; E-mail: premar51@rediffmail.comson of polygalacturonase production in the three differ-ent modes of fermentation. Effect of various agroindus-trial residues on polygalacturonase production in SSF isalso studied.Materials and MethodsMicroorganismThe actinomycete strain used in the present studywas isolated earlier in this laboratory from mangrovesediment, collected from Cochin, India and was identi-fied as Streptomyces lydicus (MTCC 7505) by IMTECH,Chandigarh, India. The culture was maintained on starchcasein agar slants at 4 °C.Preparation of inoculumThe medium used for inoculum preparation con-tained (in g/L): KH2PO4 2, K2HPO4 2, (NH4)2SO4 2,yeast extract 3 and pectin (SRL, India) 5 (12). The pH ofthe medium was adjusted to 7.0 using 1 M NaOH. Thestrain was grown for 24 h in 250-mL Erlenmeyer flaskcontaining 100 mL of liquid medium. Adequate aerationwas provided by agitation at 175 rpm at 30 °C. The ino-culum contained 2.5·104 CFU/mL.Submerged fermentationThe composition of fermentation medium was simi-lar to the medium used for the preparation of inoculumwhere pectin was replaced by wheat bran. Sterilizationwas performed at 1.05 kg/cm2 for 15 min. Fermentationwas carried out in 250-mL Erlenmeyer flask containing100 mL of growth medium with 10 % inoculum at 30°C, under static and shaking (175 rpm) conditions for 5days. Samples were collected at regular intervals andcentrifuged at 10 000 rpm for 20 min at 4 °C. The cul-ture supernatant was used as the enzyme source.Solid-state fermentationWheat bran was used as the solid substrate for SSF.A mass of 10 g of substrate was added to 250-mL Er-lenmeyer flasks and moistened with 5 mL of salt so-lution containing (in g/L): KH2PO4 4, K2HPO4 4 and(NH4)2SO4 4. The pH of the solution was adjusted to 7.0.The medium was sterilized at 1.05 kg/cm2 for 45 min,inoculated with 5 mL of broth culture and incubated at30 °C, under static and shaking (175 rpm) conditions for4 days. The final moisture content of the flask was 50 %.Slurry-state fermentationWheat bran was the substrate for SLSF too. A massof 10 g of substrate was added to 250-mL Erlenmeyerflask and moistened with 45 mL of distilled water and 5mL of salt solution containing (in g/L): KH2PO4 4,K2HPO4 4 and (NH4)2SO4 4. The pH of the solution wasadjusted to 7.0. The medium was sterilized at 1.05kg/cm2 for 45 min, inoculated with 5 mL of broth cul-ture and incubated at 30 °C, under static and shaking(175 rpm) conditions for 4 days. The final moisture con-tent of the flask was 85 %.Enzyme extractionA suitable amount of the fermented matter of SLSFand SSF was thoroughly mixed with 10 mL of distilledwater by keeping the flasks on a rotary shaker for 1 h at200 rpm. The mixture was filtered through muslin clothand the filtrate was centrifuged at 10 000 rpm for 20 minat 4 °C. The supernatant was taken for analysis.Selection of substrate for SSFVarious agroindustrial residues, coconut oil cake(COC), cotton seed oil cake (CSOC), ground nut oil cake(GOC), sesame oil cake (SOC), rice bran (RB) and wheatbran (WB), were tried as substrates for SSF. All the sub-strates were used after drying.Enzyme assaysPolygalacturonase (PG) activity was determined ac-cording to Honda et al. (13). Reaction mixture containing0.5 mL of suitably diluted enzyme was incubated with0.5 mL of 0.5 % polygalacturonic acid (SIGMA) in 100mM McIlvaine buffer (pH=7.0) for 1 h at 45 °C. The re-action was arrested by adding 2 mL of 100 mM boratebuffer (pH=9.0) followed by 1 mL of 1 % cyanoacetamide.The mixture was immersed in a boiling water bath for10 min, cooled and read against a suitable blank at 276nm using a spectrophotometer (Shimadzu, UV-2401). Oneunit (U) of PG was defined as 1 µmol of galacturonicacid released/(mL·min).Pectin lyase (PL) activity was estimated by monitor-ing the increase in absorbance at 235 nm due to the forma-tion of double conjugate bond of the unsaturated uro-nide formed during the reaction. Culture supernatant (0.3mL) was mixed with 3 mL of 1 % citric pectin (SIGMA)in 100 mM McIlvaine buffer (pH=7.0) and 2.7 mL of 100mM McIlvaine buffer (pH=7.0) and incubated for 1 h at45 °C and 150 rpm. To stop the reaction, one mL of theabove mixture was transferred to 5 mL of 0.5 M HCland read against a suitable blank at 235 nm. One unit(U) of enzyme was defined as the amount of enzyme re-quired to liberate 1 µmol of unsaturated galacturonide/(mL·min). The molar absorption coefficient used was 5500M–1·cm–1 (14).The amount of pectin esterase (PE) was determinedby titrimetric method (10). A volume of 2 mL of enzymewas added to 10 mL of 0.5 % pectin (SIGMA) in 0.1 MNaCl, the pH was adjusted to 6.0 with 0.1 M NaOH andthe mixture was incubated at 45 °C for 1 h. The amountof carboxyl groups released was determined by titrationwith 0.02 M NaOH. One unit (U) of PE was defined asthe amount of enzyme releasing one milliequivalent ofester hydrolysed/(mL·min).Enzyme yield was expressed as specific activity(U/mg protein).Total soluble protein and dry massTotal soluble protein in the culture filtrate was esti-mated according to Lowry’s method (15) using bovineserum albumin as the standard. A known mass of thefermented matter of SSF and SLSF was dried in ovenuntil constant mass and dry mass was calculated.264 N. JACOB and P. PREMA: Pectinases from Actinomycetes, Food Technol. Biotechnol. 44 (2) 263–267 (2006)Results and DiscussionPectinolytic enzyme profilePectinolytic enzyme profile of Streptomyces lydicus wasexamined under submerged fermentation for 4 days ona rotary shaker at 175 rpm and 30 °C. Different pectino-lytic activities exhibited by the culture were PG and PL.PE production was not detected in the culture superna-tant. The time course study was conducted in SmF tomonitor the production pattern of PG and PL. Fig. 1 il-lustrates the time course of PG in SmF.PG titer was maximized after 96 h of incubation inSmF. The highest enzyme activity recorded was 0.98U/mL with a specific activity of 2.39 U/mg. Though theoptimum incubation time was 96 h, specific activitieswere 51 and 71 % at 48 and 72 h, respectively. The timecourse of pectin lyase in SmF is given in Fig. 2.Pectin lyase activity reached its peak after 48 h ofincubation and then decreased. The highest enzyme titerrecorded was 0.4 U/mL having a specific activity of 2.4U/mg. Since there was no significant production of PLwhen compared to earlier reports (16), further studieswere focused on production of polygalacturonase only.Influence of mode of fermentationInfluence of mode of fermentation on the produc-tion of PG was examined using SSF, SmF and SLSF,each under static and shaking conditions. PG activitywas recorded only in SSF and SmF. SLSF was devoid ofPG activity.The highest enzyme yield was registered with SSFunder static condition. The activity was 108.5 U/g witha specific activity of 5.43 U/mg. When agitation hadbeen provided in SSF, the specific activity was decreasedby 60 %. The least activity was observed in SmF withagitation.The specific activity in SSF (static) was twofold whencompared to SmF (shaking). PE and PG production byAspergillus niger was about four and six times higher, re-spectively, in SSF than those observed in the SmF sys-tem (10). The time course of PG in SSF (static) is givenin Fig. 3.Incubation period for maximum enzyme titer was72 h in SSF, while the same was 96 h in SmF. In As-pergillus niger maximum production of PE and PL wasafter 48 h in SmF, whereas in SSF system, higher valueswere achieved in shorter time (24 h) (10). There are phy-siological differences between SSF and SmF, which inturn affect enzyme production in each fermentation sys-tem. SSF includes the growth and metabolism of micro-organisms in the absence of or near-absence of any freeflowing water. Microbial growth and product formationoccur at or near the surface of the solid substrate parti-cle. Such a system, being closer to the natural habitats ofmicrobes, may prove more efficient in producing certainenzymes and metabolites (17).SLSF was not suitable for PG production. In prac-tice, SLSF system resembles SSF except in the high mois-ture content. Very high moisture content has negativeconsequences on growth, as porosity of the medium andoxygen diffusion in the solid material is reduced (18).265N. JACOB and P. PREMA: Pectinases from Actinomycetes, Food Technol. Biotechnol. 44 (2) 263–267 (2006)0 24 48 72 96 1200.40.50.60.70.80.91.0Fermentation time / h0.00.51.01.52.02.53.0Specificactivity/ (U/mg)Enzymeactivity/ (U/mL)Fig. 1. Time course of polygalacturonase in SmF. () Enzymeactivity (U/mL), () specific activity (U/mg protein)Enzymeactivity/ (U/mL)0 12 24 36 48 60 720.000.050.100.150.200.250.300.350.400.450.00.51.01.52.02.53.0Specificactivity/ (U/mg)Fermentation time / hFig. 2. Time course of pectin lyase in SmF. () Enzyme activity(U/mL), () specific activity (U/mg protein)0 24 48 72 960204060801001200246810Specificactivity/ (U/mg)Fermentation time / hEnzymeactivity/ (U/g)Fig. 3. Time course of polygalacturonase in SSF. () Enzyme ac-tivity (U/g), () specific activity (U/mg protein)In SmF and SSF, static fermentation was found to bebetter than shaking mode. This observation may be at-tributed to the fact that actinomycetes are filamentous innature and agitation may break their mycelia.Based on the above results SSF was selected as themode of fermentation for further experiments to enhancePG yield.Substrate selection for SSFThe ideal solid substrate is one that provides all thenecessary nutrients for the microorganism. Thus the se-lection of an appropriate solid substrate plays an impor-tant role in the development of an efficient SSF process(19). Agroindustrial residues are generally considered asthe best substrate for SSF processes. The selection of asubstrate for enzyme production in an SSF process de-pends on several factors like cost and availability of thesubstrate, and thus involves screening of several agro-industrial residues. The solid substrate not only pro-vides nutrients for microbial growth but also serves as asupport for the cells (20). Substrate selection for SSF wascarried out using various agroindustrial residues (Fig.4).Among the different substrates tried, wheat branwas found to be the best for PG production. The resultis significant since wheat bran is produced worldwidein enormous quantities as an important by-product ofthe cereal industry. Enzyme production using wheat branwill make the process economical.Native PAGE of the enzyme obtained from both SmFand SSF was performed to compare the pattern of pro-tein expression in the two different modes of fermenta-tion (Fig. 5).Production of pectinases from actinomycetes is sel-dom reported (5,21). In the present investigation, it wasfound that the filamentous bacteria are a promisingsource for the same. Further optimization studies for PGproduction by Streptomyces lydicus are going on.ConclusionsThe novel strain of Streptomyces lydicus produced sig-nificant titer of PG and traces of PL in submerged fer-mentation. The strain could not produce PE. PG produc-tion reached its peak after 96 h of incubation, while PLactivity reached its maximum after 48 h. Further studieswere focused on PG and among the three modes of fer-mentation tried, the best mode of fermentation for PGproduction was SSF under static condition. The highestenzyme production was obtained after 72 h in SSF. Thespecific activity in SSF registered twofold increase whencompared to SmF. Shorter period of incubation and en-hanced enzyme production favoured SSF over SmF. PGproduction was not recorded in SLSF. The best substratefor PG production by SSF was wheat bran.AcknowledgementsThe authors thank the Director of the Regional Re-search Laboratory, Trivandrum, for providing labora-tory facilities. Financial support provided by Council ofScientific and Industrial Research, New Delhi (India),for carrying out this study is duly acknowledged.References1. N.G. Sathyanarayana, T. Panda, Purification and biochem-ical properties of microbial pectinases – A review, ProcessBiochem. 38 (2003) 987–996.2. E.F. Torres, C. Aguilar, J.C.C. Esquivel, G.V. Gonzalez: Pec-tinases. In: Enzyme Technology, A. Pandey, C. Webb, C.R.Soccol, C. Larroche (Eds.), Asiatech Publishers Inc., NewDelhi, India (2005) pp. 273–296.3. R.S. Jayani, S. Saxena, R. Gupta, Microbial pectinolytic en-zymes: A review, Process Biochem. 40 (2005) 2931–2944.4. D.R. Kashyap, P.K. Vohra, S. Chopra, R. Tewari, Applica-tions of pectinases in the commercial sector: A review, Bio-resour. Technol. 77 (2000) 215–227.5. Q.K. Beg, B. Bhushan, G.S. Hoondal, Production and char-acterization of thermostable xylanase and pectinase fromStreptomyces sp. QG-11-3, J. Ind. Microbiol. Biotechnol. 24 (2000)396–402.6. G. Kaur, S. Kumar, T. 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Influence of Mode of Fermentation on Production of Polygalacturonase by a Novel Strain of Streptomyces lydicus

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