production of extracellular phospholipase c by species...

ECOLOGIA BALKANICA2019, Vol. 11, Issue 2 December 2019 pp. 279-290

Production of Extracellular Phospholipase C by Species ofGenus Bacillus with Potential for Bioremediation

Yordan Stefanov*, Ivan Iliev, Mariana Marhova, Sonya Kostadinova

University of Plovdiv, Faculty of Biology, Department of Biochemistry and Microbiology,24 Tzar Assen Str., 4000, Plovdiv, BULGARIA

*Corresponding author: [email protected]

Abstract. The application of phospholipases produced by species of genus Bacillus in differentindustries reduces the negative impact on the environment by reducing the need of toxic chemicals,consumed energy, and produced carbon emissions. In the current study one hundred sixty-sixbacterial strains belonging to the genus Bacillus were tested for phospholipase C production.Eighty-seven percent of the studied strains demonstrated phospholipase C activity on egg-yolkagar. Strain Bacillus thuringiensis was selected as the most promising for the phospholipaseproduction with initial activity of 19.61 U/ml. The nutrient medium composition and cultivatingconditions were optimized for achieving higher enzyme yields. The highest phospholipaseproduction was achieved on the following conditions of the liquid medium: 1% of yeast extract as asource of nitrogen; 0.5% NaCl; 0.4% of glucose as a carbon source; NaHCO3 – 3 g/l; Na2HPO4 – 0.4g/l; 1 mM ZnCl2; pH 7; inoculated with 3% (1.4 x 109 cfu/ml) subculture and 8 hours duration ofthe cultivation. The production of phospholipase C by the selected strain was scaled up in abioreactor with a volume of 2l.

Key words: phospholipase C, Bacillus, bioremediation.

Introduction The human civilization is facing a global

environmental crisis, that could change thelives of everyone. A lot of different measuresare being taken to avoid furtherenvironmental problems, but the mainresponsibility lies on the global industry. Atthe moment some of the older industrialproduction technologies are being replacedwith green alternatives, based on greenchemistry and green biotechnology. Themain goal is the reduction of used dangerouschemicals and materials, reduction of theused water and energy, eliminating theproduced dangerous byproducts and toreduce the production of greenhouse gases.

One of the possible solutions to theseproblems is the use of biocatalysts –enzymes, in different kinds of chemicalprocesses. They can effectively increase thespeed of the occurring chemical reactions,they are active at a lower temperature,which leads to a reduction in the consumedenergy for heating, they reduce the quantityof the used toxic chemical compounds andthey can be produced from by-products andwastes of other industries. For theproduction of enzymes, bacterial species areoften preferred. Of them Bacillus spp. havemany favorable properties. Some of thespecies have GRAS status - they areconsidered as safe for food and food

© Ecologia Balkanicahttp://eb.bio.uni-plovdiv.bg

Union of Scientists in Bulgaria – PlovdivUniversity of Plovdiv Publishing House

Production of Extracellular Phospholipase C by Species of Genus Bacillus with Potential for Bioremediation

additives production. They have shortdivision cycles, they can be cultured in shortperiods, they can secrete the desired proteinsin the medium, which makes the enzymeeasier for purification. All these propertiesare making production cost-effective(SEWALT et al., 2016).

In the food and beverage productionindustries enzymes also find broad field offapplication. For example, phospholipasescan be used in the refining process ofvegetable oils. The biological catalyzers canbe used as a replacement to the classicalrefining technologies, which are relying on acaustic process using strong alkaline andphosphoric acid solutions for the removal ofphospholipids, which reduce the quality ofthe oils. Data form industrial experiments atBunge's oil refining plants (USA) show, thatthe used enzymes for degumming havereduced the amount of consumed energyand the produced greenhouse gases. For therefining of 266 000 tons of soy oil theconsumed energy is reduced with 112 000GJ, the carbon emissions are reduced with 12000 tons, SO2 emissions are reduced with 140tons, PO4 emissions are reduced with 100tons, and the produced ethylene is with 4tons less (DE MARIA et al., 2007). On anannual basis, the food industry throws awaytons of materials rich in keratin in the formof hair, fur, skin, nails, and other by-products which are considered dangerouswaste, but they could be a good source ofamino acids. It is possible these wastematerials to be used as a substrate forenzymatic hydrolysis process usingproteolytic enzymes. The final products willbe rich in single amino acids and smallpeptides, which could be used as a valuablenutrition source for animals (SINGH & BAJAJ,2017). Another valuable application is in thebaking industry. The quality of the producedbread and similar products is declining intime with the crystallization of the starch inthe products. In this case, amylase enzymescan be used to increase the shelf life and thequality of the products. Careful calculationsshow that the financial loses of producing

bread with short shelf life are bigger than thecost of the treatment of the drought withamylases (JEGANNATHAN & NIELSEN, 2013).

But enzymes can be used not only inmanufacturing processes. They can be usedfor the treatment of chemical contaminationsin the environment. CHRISTOVA et al. (2019)have identified a strain of Bacillus cereus,which has the ability to degrade up to 93%of the oil hydrocarbons in the examinedmedium for about 48 hours. The researchgroup has immobilized the isolated strainand their results show that the preparationremains almost fully active for 47 days, at28°C up to 20 cycles of usage. Other authorsalso have reported similar results.SAKTHIPRIYA et al. (2015) have isolated strainof Bacillus cereus, which can degrade up to80% of the oil hydrocarbons in thecontaminated environment. Enzymes can beused also for the removal of toxicagrochemical contaminants in the soil, likepesticides, herbicides, and fungicides. Somestrains of Bacillus amyloliquefaciens have hy-1gene with a length of 858 bp, which is codingprotein, which can hydrolyze the fungicidecarbendazim to 2-aminobenzimidazole. Theisolated protein is identified as a type ofphospholipase, which successfully canremove the fungicide off the surface ofcontaminated cucumbers. This experimentproves that enzymes can be used as aneffective tool in the bioremediation processes(LI et al., 2019). There are already developedtechnologies that use Bacillus subtilis andother species that produce highly activelipase and phospholipase enzymes thathydrolyze contaminated with fats and oilswaste waters. The produced free fatty acidsand alcohols can then be easily digest by theactive sludge in the water treatment plant(Patent No. PCT/JP2013/055504).

Although lipases and phospholipasesare hydrolytic enzymes in nature, they couldbe used in valuable biosynthesis industrialprocesses. Their ability to carry ontransesterification reactions can be used forthe biosynthesis of fatty acids alkyl esters,which commercially are better known as

280

Stefanov et al.

biodiesels – an important source ofrenewable fuels (ANOBOM et al., 2014).

The aim of the current work is toexamine the extracellular phospholipase Cproduction capabilities of strains of genusBacillus and to optimize the condition for theenzyme production.

Materials and MethodsBacterial strainsStrains of genus Bacillus (166 strains) from

the microbial collection of the Department of“Biochemistry and microbiology", Faculty ofBiology, University of Plovdiv, Bulgaria wereexamined for phospholipase C activity.Cultures of the organisms were maintained onnutrient agar medium at 4°С for routinelaboratory use. For long-term use, the strainswere maintained in nutrient agar underparaffin layer at 4°С.

The initial screening was based on theability of the strains to hydrolyzephospholipids in egg yolk agar medium.

Hydrolysis of phospholipidsEach strain was inoculated on egg yolk

agar (one egg yolk; nutrient broth – 3 g;yeast extract – 0.5 g; glucose – 0.5 g; 5 ml0.1M CaCl2.2H2O; 95 ml H2O; pH 7.2) andincubated for 24 to 48 hours at 37°С. Theformed halo around the colony wasmeasured. Strains that have formed halolarger than 5 mm were taken for furtheranalyses.

Fermentation conditions and separation ofculture filtrates

A subculture was prepared prior to thefermentation by inoculation of the strain innutrient broth and incubation for 8h on arotary shaker at 37°С, 120 rpm. The celldensity of the culture was then modified to6.0 McF units.

Erlenmeyer flasks with volume of 300ml, containing 30 ml growth medium (caseinhydrolysate – 10 g/l; Bacto Peptone – 10 g/l;NaCl – 5 g/l; glucose – 4 g/l; NaHCO3 – 3 g/l; Na2HPO4 – 0.4 g/l; ZnCl2 – 0.01 g/l; pH 7;sterilized for 15 minutes at 121°С) wereinoculated with 3% (6.0 McF) subculture(GERASIMENE et al., 1980). The inoculated

flasks were set on a rotary shaker at 37°С,120 rpm for 12 - 48 hours. For the separationof the cells, 10 ml culture medium werecentrifuged at 4°С, 14 000 rpm for 20minutes. The supernatant was collected forfurther analysis.

Optimization of the culture conditions forenzyme production

Optimum phospholipase productionwas studied at different incubation periods(0 – 36 h), with different volumes of theinoculum between 1 – 10%, carbon sources(mannose, fructose, maltose and ribose),concentrations of the carbon source (0.2 –0.8%), different sources of nitrogen(bactopeptone, casein hydrolysate, yeastextract, beef extract, KNO3, (NH4)2SO4), anddifferent metal ions (Mg2+, Mn2+, Zn2+, Ca2+).

Fermentation in bioreactorFed-batch cultivation was performed in

a 2 L bench-top bioreactor (Minifors, InforsHT, Switzerland) equipped with turbineimpeller and connected to a digital controlunit. The set points for temperature was37°C, constant pH was not maintained, nocompressed air was supplied, the agitationspeed was maintained to 100 rpm, noantifoam agent was used. The compositionof the medium was identical to theestablished optimal medium composition inflasks experiments. Every 2 hours, 20 mlsample was taken from the vessel for thedetermination of the phospholipase Cactivity, cell density, and pH of the culture.

Enzyme assaysThe determination of phospholipase C

activity is based on the enzymatic digestionof L-α-Phosphatidylcholine, extraction of theproduced phosphate compounds, anddetermination of their concentrationaccording to the method described byTAKAHASHI et al. (1981).

Results and DiscussionOne hundred and sixty-six Bacillus strains

were tested for production of PLC in amedium with phosphatidylcholine as esubstrate. The number of strains by species ispresented in Fig. 1a.

281


Fig. 1. Enzyme activities of strains of genus Bacillus – qualitative analysis.166 examined strains (a.). Phospholipase C activity of strains of genus Bacillus (b.).

After the qualitative assays strains,which formed hydrolysis zones or formedhaloes larger than 5 mm, were selected forthe quantitative determination of enzymeactivity. Of all examined strains thirteenwere selected for quantitative determinationof their extracellular phospholipase Cactivity. Bacillus thuringiensis №17 hadhighest phospholipase C activity reaching19.61 U/ml (Fig. 2). In previous research wehave shown that these strains can producealso extracellular amylase and proteaseenzymes with activity within the range of2.80 – 9.20 U/ml (STEFANOV et al., 2018a;STEFANOV et al., 2018b).

Fig. 2. Quantitative determination ofphospholipase C activity of strains of genus

Bacillus

The present work and in previousstudies on protease (STEFANOV et al., 2018a)and amylase (STEFANOV et al., 2018b) activityshowed that the examined strains arecharacterized by a diverse extracellularenzyme production. From all Bacillus cereusstrains, 94% produced phospholipase C, and93% produced proteases. Only 29% couldproduce lipases, 27% amylase enzymes, and3% had cellulolytic activity. Eighty-sevenpercent of all examined strains of Bacillusthuringiensis had phospholipase C activity,88% had protease activity, 16% had amylaseactivity, 15% had lipase activity, and only 2%exhibited cellulolytic activity. All examinedstrains of Bacillus sphaericus exhibited weakextracellular enzyme activity.

Bacillus species are known for theirability to secrete a vast variety of proteins inthe culture medium. They can produce morethan 40 different extracellular enzymes.Transcriptomic analyses of the speciesduring different stages of cultivation showthat they produce more than 3800 codingtranscripts but in varying quantity. It hasbeen shown that Bacillus pumilus transcribesgenes that are coding two lysophospho-lipases and three phospholipases. Theirexpression is initiated during theexponential phase of growth, but itsmaximum is reached during the earlystationary phase of growth. Some species of

282

Stefanov et al.

Bacillus have several genes that codedifferent types of extracellular proteases,which are form bio-technical interest. It isshown that the expression of epr and subEbegins at the exponential phase of growth,while aprE, aprX, and wprA are highlyexpressed during the stationary phase ofgrowth. The major proteolytic activity of theisolated supernatant is contributed to theproducts of these 3 genes. All this shows thatthe expression of the different proteases iscontrolled by different regulatorymechanisms and may even have differentsecretion paths (HAN et al., 2017).

The production of a vast variety ofextracellular enzymes makes some strains ofgenus Bacillus valuable tools for saving theenvironment and avoiding ecologicaldisasters. It has been shown that strains thatproduce lipolytic, proteolytic, and/oramylolytic enzymes could efficiently reducethe biological oxidation demand (BOD),chemical oxidation demand (BOD), nitratesand, phosphates by up to 56.25% after 72hours (SONUNE & GARODE, 2018). This couldprevent the depletion of oxygen in the waterand the suffocation of its main consumers.

Chromatographic analysis of the productsfrom the hydrolysis reactions

The identification of the products of thehydrolysis reaction of L-α- phosphatidylcholine(Sigma-Aldrich, USA), mediated byphospholipase C, isolated from Bacillusthuringiensis №17, was done using thin-layerchromatography (Fig. 3). The reduced amount ofthe substrate (Fig. 3b) correlates with theappearance of the reaction products (Fig. 3a). Itcan be seen from the chromatogram thatphospholipase C from B. thuringiensis №17hydrolyzed the highest amount of the substrate.

The used substrate for the hydrolysisreaction was sonicated as a pretreatment toform lipid vesicles. Phospholipase enzymesprefer phospholipid substrates in the form ofsmall vesicles with a diameter of 20-100 nminstead of single molecules. It wasestablished by other researchers that theproduced phospholipases from species ofgenus Bacillus bind strongly to vesicles with

a high content of phosphatidylcholine.Although the strong binding between thesubstrate and the enzymes, which lasts foran average of 378 ± 49 ms, the highphosphatidylcholine concentration reducesthe enzyme activity. Phospholipases alsoprefer smaller vesicles with higher surfacetension (YANG et al., 2015).

Fig. 3. Thin-layer chromatography of thesubstrate and the products of the enzyme

hydrolysis with phospholipase C from B. thuringiensis №17. (1) L-α-

phosphatidylcholine (99%, Sigma-Aldrich,USA); (2) the substrate after hydrolysis withPLC from B. thuringiensis №17. System (a) -

petroleum ether:diethyl ether:acetic acid(6:4:0.2); System (b) -

chloroform:methanol:water (65:25:4).

Dynamics of the phospholipase productionduring growth

The production of phospholipaseenzymes started after the second hour of

283


cultivation and it slowly increased, reachingits maximum at the 8-th hour with anactivity of 20.69 U/ml. After twelve hours ofcultivation, the enzyme production slowlydropped reaching 2.42 U/ml at the 24-thhour. The pH of the culture medium doesn’tchange dramatically during the cultivation.In the beginning, the medium was weaklyacidified to pH 6.75 at the fourth hour andthen the medium gradually was alkalized topH 7.90 at the 24-th hour. The analysis of thecell density showed that the exponentialphase of growth begins nearly at the beggingof the cultivation and it is over at the 8-thhour (Fig. 4).

The maximum enzyme production wasreached between the late exponential phaseof growth and the beginning of thestationary phase. The rate of phospholipaseproduction correlates with cell density. This

type of fermentation, in whichphospholipase C is produced from speciesof genus Bacillus is classified as growthassociates (SHILOACH et al., 1973). Thisphenomenon may be explained by the factthat many (primary) enzymes, producedfrom Bacillus, are needed for the survivaland development of the bacterial cultureespecially in the early stages of thefermentation (BLANCO et al., 2016). It hasbeen discovered that the synthesis ofphospholipases is under the control of thequorum-sensing system of the bacterialcommunity (ELLEBOUDY et al., 2011; DONG

et al., 2002), but the presence of theseenzymes is not due to cell lysis orsporulation. Thus, higher phospholipaseproduction is achieved at certain celldensity. The enzymes are secreted in themedium (ELLEBOUDY et al., 2011).

Fig. 4. Growth and phospholipase C activity of Bacillus thuringiensisstrain 17 in a liquid medium of GERASIMENE et al. (1980).

Effects of different carbon sourcesTo determine the effects of different

carbon sources on the phospholipase Cproduction, glucose, fructose, ribose, inulin,or maltose, respectively, at a concentration of0.4%. were added to the base medium. Thecontrol medium contained glucose as a

source of carbon (Fig. 5). Our results showedthat maximum enzyme production wasachieved when the medium wassupplemented with glucose. High enzymeactivity was also achieved when the strainwas incubated in medium, containingmaltose. The production was reduced when

284

Stefanov et al.

the medium contained ribose or inulin (Fig.5). The results of THAYUMANAVAN &BOOPATHY (2005) also show that glucose is agood choice as a source of carbon for theproduction of phospholipase C from B.thuringiensis. Bacillus thuringiensis can utilizeother carbohydrates like fructose, sucrose,and lactose, but more complex carbon sources(like starch) are less preferable. This canreduce the cell density and the production ofextracellular enzymes respectively (BULLA etal., 1980). It has been discovered that the useof molasses as a cheaper source of carbonresults in lower phospholipase production(THAYUMANAVAN & BOOPATHY, 2005).

Fig. 5. Effect of the carbon source on thephospholipase C production.

*Control – glucose.

According to ZHAO et al. (2018), researchhigh concentration of the glucose, fructose ormaltose in the liquid medium may reduce oreven inhibit the synthesis of phospholipaseenzymes. Our results showed that a gradualincrease in the concentration of glucose to amaximum amount of 0.4% leads to a rise inphospholipase C production. Further increasein the concentration results in a decrease andinhibition of the enzyme synthesis. Theresults of THAYUMANAVAN & BOOPATHY(2005), show that maximum phospholipaseproduction is achieved when the mediumcontained 0.65% of glucose. For manymicroorganisms, glucose is the preferredcarbon source. When it is present in the

medium in excess, it may repress thecatabolism of other substrates and thesynthesis of the needed enzymes. Thisphenomenon is called catabolic repressionand it is an evolutionary mechanism, whichallows the organisms to utilize first thesubstrate, which guarantees them the fastestgrowth in the competitive environment(SINGH & BAJAJ, 2004).

Fig. 6. Effect of glucose concentrations on thephospholipase C production.

*Control - 0.4% glucose.

Effects of different nitrogen sourcesThe source of nitrogen is another

important factor for the growth of thebacterial culture and for the production ofphospholipase C. In the current study theinfluence of six nitrogen sources was studied– peptone, bactopeptone, casein hydrolysate,yeast extract, potassium nitrate, andammonium sulfate. The results showed thatthe production of phospholipase C is slightlyhigher when the growth medium wassupplemented with yeast extract. It has beenproven that Bacillus thuringiensis №17 cannotutilize inorganic sources of nitrogen (Fig. 7).Although some strain can utilize inorganicnitrogen compounds, the majority of Bacillusthuringiensis strains prefer organic nitrogen(THAYUMANAVAN & BOOPATHY, 2005; EL‐BENDARY, 2006). To ensure the growth of thebacterial culture the medium must besupplemented with source of at least one ofthe following amino acids – glutamate,

285


aspartate, valine, leucine, serine orthreonine, but the addition of cysteine orcystine will lead to full growth inhibitionand the formation of toxic compounds (EL‐BENDARY, 2006), but complex organicsources of nitrogen are preferred – peptone,casein hydrolysate, yeast extract and others(ICGEN et al., 2002). When the medium is richin free proteins and protein hydrolyzates theadded free amino acids are used as a sourceof carbon (EL‐BENDARY, 2006).

Fig. 7. Effect of nitrogen sources on thephospholipase C.

*Control - the original medium as describedby GERASIMENE et al. (1980).

Effects of different metal ionsМore than half of the discovered

enzymes are metalloenzymes, including thephospholipases, but according to otherresearchers, only 30% of the enzymes requiremetal ions to carry out their normalfunctions (WALDRON & ROBINSON, 2009).Phospholipases, produced by species ofgenus Bacillus contain two zinc atoms intheir protein structure. The removal of oneof the zinc atoms results in partial reductionof the enzyme activity. The removal of thesecond atom results in the full inhibition ofthe activity. This effect is reversible if zincions are added to the enzyme solution(LITTLE & OTNASS, 1975). Some metal ionscan enhance the enzyme activity and otherscan inhibit it. In the current study, theinfluence of 4 sources of metal ions in 1 mM

concentration (CaCl2, ZnCl2, MgCl2, MnCl2)on the phospholipase C production wasstudied. As a control was used a mediumthat didn’t contain any metal ions. Theresults showed that the enzyme productionis reduced when the medium containedmanganese ions. The production ofphospholipase C is greatly enhanced whenthe medium contained zinc ions. Otherresearchers also report that the presence ofmetal ions in the medium is of greatimportance for the growth of the bacterialproducer and for the enzyme production(THAYUMANAVAN & BOOPATHY, 2005). Zincions can directly participate in the enzymereaction or they may maintain the proteinstructure stable. They also function in allcatalytic sites as Lewis acid – chemicalcompound, containing free orbital, which iscapable of accepting electron pairs.

Fig. 8. Effect of metal ions on thephospholipase C activity of B. thuringiensis.

*Control - medium with no metal ionsupplementation.

Effects of the volume of inoculumTo determine the effects of the volume

of the inoculum over the production ofphospholipase C, Erlenmeyer flasks with asterile medium where inoculated withvarying volumes (between 1 – 10%) of 8-thhour bacterial culture with a cell density of1.4 x 109 cfu/ml. It was found that maximumenzyme production occurs when themedium is inoculated with 7.5% of

286

Stefanov et al.

inoculum. The quantity of the producedenzyme between the different examinedvolumes of inoculum is not significant, butthe tendency is clearly visible. The rate ofenzyme production rises with the largervolumes of inoculum. The reduction of theenzyme production when using volumeslarger than 7.5% may be explained with themore intensive consumption of nutrients inthe medium. The bacterial culture alsoproduces compounds that are inhibiting thegrowth and development of the culture.(HAYES & LOW, 2009).

Fig. 9. Effect of inoculum concentration onthe phospholipase C production.

*Control - 3% inoculum.

Production of phospholipase C in bioreactorThe dynamics of phospholipase

production was studied in a scale-upexperiment using a bioreactor with a volumeof 2 liters. Bacillus thuringiensis №17 wasinoculated in a medium with the determinedoptimum composition. The production ofphospholipase C enzymes began at thesecond hour of cultivation, slowly increasedand reached its maximum at the 8th hour ofcultivation with activity of 14.87 U/ml afterwhich the production dropped down.During the cultivation, the medium wasslowly acidifying reaching pH 6.21 at thetwelve hours of cultivation. The bacterialculture entered almost immediately in theexponential phase of growth, which endedafter the 6-th hour of cultivation. In our

scale-up experiment, maximum phospho-lipase production was achieved in the earlystationary phase of cultivation with pH 6.17of the medium.

Fig. 10. Dynamics of the phospholipaseproduction using bioreactor with volume of

2 liters.

Although during the cultivation inbioreactor and Erlenmeyer flasks theproduction of phospholipase C was inidentical time intervals, the cell density, pHof the medium and enzyme activity varied.During the cultivation in flasks, the mediumfirst slowly acidified to pH 6.75, but later themedium was alkalized to pH 8. During thecultivation in a bioreactor, the medium wassteadily acidified to pH 6. The difference inthe cell density of the two cultures was quitesignificant. In flask maximum enzymeproduction was achieved when the culturehad a cell density of 7.4 McF, but inbioreactor only 5.9 McF. The differenceequals 0.5 x 109 cfu/ml. Even the enzymeactivity was lower when the strain wascultivated in a bioreactor. This can beexplained with many factors that areimportant during scale-up fermentation –the form of the vessel, the speed of agitation,the supplementation with oxygen,production of foam, the influence ofantifoaming agents and others. Manyresearchers also describe similar effects –although using similar medium compositionand fermentation conditions the results

287


between flask and bioreactor fermentationdiffer from one another (BEG et al., 2003).The lower cell density may correlate withthe lower enzyme production. It isdiscovered that cell communication in theform of quorum sensing is regulating thegene expression in the bacterial community.In gram-positive bacteria, cellcommunication is using cytoplasmicsensors, regulated by secreted andreimported signal peptides. Quorumsensors in genus Bacillus include Rap,NprR, and PlcR, which are part of the RNPPprotein family. Besides Rap these RNPPproteins are transcriptional factors, whichdirectly regulate the gene expression. In thisway, quorum-sensing regulates manyimportant functions in the Bacillus cereusgroup. In this situation, quorum-sensingregulates the expression of phospholipaseenzymes (which expression is regulated byPlcR) (SLAMTI et al., 2014). If the cell densityis not high enough, the cell to cellcommunication is hampered and theexpression of some key enzymes is reduced.

ConclusionIt was determined that B. thuringiensis

№17 was the best producer of phospho-lipases between the studied species of genusBacillus, with an activity of 19.61 U/ml. Thestrain showed consistent extracellularphospholipase C activity. The production ofphospholipase C was detected at the secondhour of cultivation and it reached itsmaximum at the 8-th hour of cultivationduring the end of the exponential and thebeginning of the stationary phase of growth.Such early production of extracellularlipolytic enzymes is a feature of economicvalue. The highest phospholipase Cproduction was achieved when the mediumwas supplemented with 0.4% of glucose,yeast extract as a source of nitrogen and 1mM ZnCl2 and inoculated with 7.5% ofinoculum. After the optimization of themedium, the duration of the cultivation, andthe volume of used inoculum, the activityreached 31 U/ml. Scale-up study with a

benchtop bioreactor with a volume of 2 litersachieved the highest exoenzyme productionat the 8 hours of cultivation, whichcorrelated with the flask experiments. Theselected strain and studied phospholipaseenzyme have a significant potential forapplication in the field of bioremediation –cleaning contaminated soils, digesting ofspilled oils and fats in wastewaters and evenreducing the energy consumption and thegenerated greenhouse gas emissions in someindustries. Replacing the old chemicaltechnology of refining oils with enzymedegumming using phospholipases results inreduced production of carbon emissions,reduced consumption of dangerous chemicalcompounds and product with higherquality. Phospholipases can be a sustainableand effective alternative to some old andinefficient technologies.

ReferencesANOBOM C.D., А.S. PINHEIRO, R.A. DE-

ANDRADE. 2014. From structure tocatalysis: Recent developments in thebiotechnological applications oflipases. - BioMed Research International,2014: 1–11.

BEG Q.K., V. SAHAI, R. GUPTA. 2003.Statistical media optimization andalkaline protease production fromBacillus mojavensis in a bioreactor. -Process Biochemistry, 39(2): 203-209.

BLANCO A.S., O.P. DURIVE, S.B. PÉREZ, Z.D.MONTES. 2016. Simultaneousproduction of amylases and proteasesby Bacillus subtilis in brewery wastes. -Brazilian Journal of Microbiology, 47: 665-674.

BULLA L.A., D.B. BECHTEL, K.J. KRAMER, Y.I.SHETHNA. 1980. Ultrastructure,physiology and biochemistry ofBacillus thuringiensis. - CRC CriticalReviews in Microbiology, 8: 147–203.

CHRISTOVA N., L. KABAIVANOVA, L.NACHEVA, P. PETROV, I. STOINEVA.2019. Biodegradation of crude oilhydrocarbons by a newly isolatedbiosurfactant producing strain. -

288

Stefanov et al.

Biotechnology & BiotechnologicalEquipment,, 33(1): 863-872. [DOI].

CLAUSEN I.G., S.A. PATKAR, K. BORCH, T.HALKIER, M. BARFOED, K. CLAUSEN,C.C. FUGLSANG, L. DYBDAL. 1998.Novoenzyme A/S Denmark. PCTInternational Patent applicationWO1998/26057.

DE MARIA L., J. VIND, K.M. OXENBØLL, A.SVENDSEN, S. PATKAR. 2007.Phospholipases and their industrialapplications. - Applied Microbiology andBiotechnology, 74:290–300.

DONG Y.H., A.R. GUSTI, Q. ZHANG, J.L. XU,L.H. ZHANG. 2002. Identification ofQuorum-Quenching N-AcylHomoserine Lactonases from Bacillusspecies. - Applied and EnvironmentalMicrobiology, 68: 1754-1759.

DURBAN M.A., J. SILBERSACK, T. SCHWEDER,F. SCHAUER, U.T. BORNSCHEUER. 2006.High level expression of a recombinantphospholipase C from Bacillus cereus inBacillus subtilis. - Applied Microbiologyand Biotechnology, 74(3): 634–639.

EL‐BENDARY M.A. 2006. Bacillus thuringiensisand Bacillus sphaericus biopesticidesproduction. - Journal of BasicMicrobiology, 46: 158-170.

ELLEBOUDY N.S.H., M. ABOULWAFA, N.A.HASSOUNA. 2011. Characterization ofPhospholipase C Productivity byPseudomonas aeruginosa, Bacillus cereusand Staphylococcus aureus isolates. - TheJournal of American Science, 7: 545-566.

EXTON J.H. 1997. Phospholipase D:enzymology, mechanisms ofregulation, and function. - PhysicalReview, 77(2): 303-320.

GERASIMENE G.B., I.P. MAKARIUNAITE, V.V.KULENE, A.A. GLEMZHA. 1980.Biosynthesis of extracellularphospholipase C (lecithinase) fromBacillus cereus depending on thenutrient medium composition and pH.- Prikladnaia Biokhimiia i Mikrobiologiia,16(4): 523-527

HAN L.L., H.H. SHAO, Y.C. LIU. 2017.Transcriptome profiling analysis reveals

metabolic changes across various growthphases in Bacillus pumilus BA06. - BMCMicrobiology, 17(1): 156.

HAYES C.S., D.A. LOW. 2009. Signals ofgrowth regulation in bacteria. - CurrentOpinion in Microbiology, 12(6): 667–673.

ICGEN Y., B. ICGEN, G. OZCENGIZ. 2002.Regulation of crystal proteinbiosynthesis by Bacillus thuringiensis: II.Effects of carbon and nitrogen sources.- Research in Microbiology, 153, 605–609.

JEGANNATHAN K.R., P.H. NIELSEN. 2013.Environmental assessment of enzymeuse in industrial production – aliterature review. - Journal of CleanerProduction, 42, 2013:228-240.

LI Y., M. CHI, X.Z. GE. 2019. Identification of anovel hydrolase encoded by hy-1 fromBacillus amyloliquefaciens forbioremediation of carbendazimcontaminated soil and food. -International Journal of Agricultural andBiological Engineering, 12(2): 218–224.

LITTLE C., A. OTNASS. 1975. The metal iondependence of phospholipase C fromBacillus cereus. - Enzymology, 391(2): 326-333.

NIELSEN E.W. 2004. Principles of cheeseproduction. Handbook of food andbeverage fermentation technology.Marcel Dekker, New York.

NILSSON-JOHANSSON L., U.I. BRIMBERG, G.HARALDSSON. 1988. Experience ofprerefining of vegetable oils with acids.- Fat Science Technology, 90: 447-451.

Patent PCT/JP2013/055504. 2013. Biologicaltreatment of water, waste water, or sewagecharacterised by the microorganisms used fordigestion of grease, fat, oil. Filed underPatent Cooperation Treaty (PCT).

SAKTHIPRIYA N., M. DOBLE, J.S. SANGWAI.2015. Bioremediation of Coastal andMarine Pollution due to Crude OilUsing a Microorganism Bacillus subtilis.- Procedia Engineering, 116: 213-220.

SEWALT V., D. SHANAHAN, L. GREGG, J. LAMARTA, R. CARRILLO. 2016. TheGenerally Recognized as Safe (GRAS)process for industrial microbial enzymes.- Industrial Biotechnology, 12: 295–302.

289

https://doi.org/10.1080/13102818.2019.1625725


SHILOACH J., S. BAUER, I. VLODAVSKY, Z.SELINGER. 1973. Phospholipase-C fromBacillus cereus: Production, purification,and properties. - Biotechnology andBioengineering, 15: 551-560.

SINGH S., B.K. BAJAJ. 2017. Potentialapplication spectrum of microbialproteases for clean and greenindustrial production. - Energy,Ecology and Environment, 2: 370.

SLAMTI L., S. PERCHAT, E. HUILLET, D.LERECLUS. 2014. Quorum sensing inBacillus thuringiensis is required forcompletion of a full in fectious cycle intheinsect. - Toxins, 6(8): 2239-55.

SONUNE N., A. GARODE. 2018. Isolation,characterization and identification ofextracellular enzyme producer Bacilluslicheniformis from municipalwastewater and evaluation of theirbiodegradability. - BiotechnologyResearch and Innovation, 2(1): 37-44.

STEFANOV Y., I. ILIEV, M. MARHOVA, S.KOSTADINOVA. 2018a. Isolation andPurification of Proteolytic Enzymes,Produced by Strains of Genus Bacillus.- Ecologia Balkanica, 10(2): 185-197.

STEFANOV Y., I. ILIEV, M. MARHOVA, S.KOSTADINOVA. 2018b. Optimization ofnutritive medium composition forproduction of amylase by Bacillusstrains. - Journal of Bioscience andBiotechnology, 7(2-3): 103-107.

TAKAHASHI K., J.L. CASEY, J.M. STURTEVANT.1981. Thermodynamics of the bindingof D-glucose to yeast hexokinase. -Biochemistry, 20(16): 4693-4697. [DOI]

THAYUMANAVAN P., R. BOOPATHY. 2005.Optimization of Phosphatidylinositol-specific Phospholipase C ProductionUsing Response Surface Methodology.- World Journal of Microbiology andBiotechnology, 21: 1393-1399.

VANCE J.E., D.E. VANCE. 2008. Biochemistry ofLipids, Lipoproteins and Membranes.Elsevier.

WAITE М. 1996. New ComprehensiveBiochemistry, Chapter 8 - Phospholipases.Elsevier.

WALDRON K.J., N.J. ROBINSON. 2009. How dobacterial cells ensure thatmetalloproteins get the correct metal?.- Nature Reviews Microbiology, 7: 25–35.

YANG B., M. PU, M.H. KHAN, L. FRIEDMAN, N.REUTER, F.M. ROBERTS, A. GERSHENSON.2015. Quantifying Transient Interactionsbetween Bacillus Phosphatidylinositol-Specific Phospholipase C andPhosphatidylcholine Rich Vesicles. -Journal of the American Chemical Society,137(1): 14-17.

ZHAO Y., Y. XU, F. YU. 2018. Identification of anovel phospholipase D gene and effectsof carbon sources on its expression inBacillus cereus ZY12. - Journal ofMicrobiology., 56: 264-271.

Received: 02.12.2019Accepted: 23.12.2019

© Ecologia Balkanicahttp://eb.bio.uni-plovdiv.bg

Union of Scientists in Bulgaria – PlovdivUniversity of Plovdiv Publishing House

https://doi.org/10.1021/bi00519a026

production of extracellular phospholipase c by species...

Documents