novel sequential screening and enhanced...

Research ArticleNovel Sequential Screening and Enhanced Production ofFibrinolytic Enzyme by Bacillus sp IND12 Using ResponseSurface Methodology in Solid-State Fermentation

Ponnuswamy Vijayaraghavan1 P Rajendran2 Samuel Gnana Prakash Vincent1

Arumugaperumal Arun3 Naif Abdullah Al-Dhabi4

Mariadhas Valan Arasu4 Oh Young Kwon5 and Young Ock Kim6

1Centre for Marine Science and Technology Manonmaniam Sundaranar University Rajakkamangalam Kanyakumari DistrictTamil Nadu 629 502 India2Kanyakumari Field Centre Central Marine Fisheries Research Institute Kanyakumari Tamil Nadu 629702 India3Department of Biotechnology Kalasalingam University Srivilliputtur Virudhunagar Tamil Nadu 626126 India4Department of Botany and Microbiology Addiriyah Chair for Environmental Studies College of Science King Saud UniversityP O Box 2455 Riyadh 11451 Saudi Arabia5Department of Medical Education and Medical Humanities School of Medicine Kyung Hee University Seoul Republic of Korea6Department of Medicinal Crop Research RDA Eumseong Chungbuk 369-873 Republic of Korea

Correspondence should be addressed to Ponnuswamy Vijayaraghavan venzymesgmailcomand Mariadhas Valan Arasu mvalanarasugmailcom

Received 8 August 2016 Revised 30 November 2016 Accepted 13 December 2016 Published 22 February 2017

Academic Editor Denise Freire

Copyright copy 2017 Ponnuswamy Vijayaraghavan et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Fibrinolytic enzymes have wide applications in clinical andwaste treatment Bacterial isolates were screened for fibrinolytic enzymeproducing ability by skimmed milk agar plate using bromocresol green dye fibrin plate method zymography analysis and goatblood clot lysis After these sequential screenings Bacillus sp IND12 was selected for fibrinolytic enzyme production Bacillus spIND12 effectively used cow dung for its growth and enzyme production (687plusmn65Ug substrate) Further the optimum bioprocessparameters were found out for maximum fibrinolytic enzyme production using cow dung as a low cost substrate under solid-state fermentation Two-level full-factorial experiments revealed that moisture pH sucrose peptone and MgSO4 were the vitalparameters with statistical significance (119901 lt 0001) Three factors (moisture sucrose and MgSO4) were further studied throughexperiments of central composite rotational design and response surface methodology Enzyme production of optimized mediumshowed 4143 plusmn 1231Ug material which was more than fourfold the initial enzyme production (978 plusmn 364Ug) The analysis ofvariance showed that the developed response surface model was highly significant (119901 lt 0001) The fibrinolytic enzyme digestedgoat blood clot (100) chicken skin (83 plusmn 36) egg white (100) and bovine serum albumin (29 plusmn 49)

1 Introduction

Fibrinolytic enzymes are used as thrombolytic agents totreat cardiovascular diseases and stroke The plasminogenactivator molecules namely urokinase streptokinase andtissue plasminogen activator are commonly used in throm-bolytic therapy but are expensive and also have unintendedphysiological effects like bleeding complication low fibrinspecificity and short half-lives [1] Hence the search for

the effective and safe thrombolytic agent from differentbiosources keeps growing worldwide Study reports are avail-able on fibrinolytic enzymes from various organisms includ-ing Bacillus polymyxa [2] Escherichia coli [3] Pseudomonas[4] Streptomyces [5] Paenibacillus sp IND8 [6] and Bacilluscereus IND1 [7] Fibrinolytic enzymes have the ability toinhibit blood coagulation and are able to degrade the fibrin[8] These enzymes have been reported to be produced in

HindawiBioMed Research InternationalVolume 2017 Article ID 3909657 13 pageshttpsdoiorg10115520173909657

2 BioMed Research International

both solid-state fermentation (SSF) and submerged fermen-tation processes

SSF has emerged as a useful method for production ofenzymes and many significant pharmaceutical products Ina SSF process usage of convenient substrate is one of theimportant factors and the yield is considerably higher thansubmerged fermentation [9] Agroindustrial residues likewheat bran [9] pigeon pea [10] potato peel [11] grass powder[12] and chicken feather [13] were used for the production ofenzymes Apart from these agroindustrial wastes researchersattempted to use various effluents including dairy and tan-nery industrial effluents for the production of proteolyticenzymes These industrial wastes are rich in nitrogen andcarbon sources Several studies have proved the potentialuse of these industrial wastes to minimize pollution and toincrease the product yield [14 15] The marine food wasteshrimp peptone was also shown to be a potential substratefor SSF involving the production of proteases recently [16]The agricultural wastes such as peanut shell corncob pomaceof corn and peels of cassava were used for the productionof tetracycline [17] The antibiotic Cephamycin C wasproduced in SSF using wheat straw deoiled cake of cottonseed sunflower cake and corn steep liquor [18] and wheatbran and sweet lemon peel was used as the substrate forthe production of nigerloxin in SSF [19] Apple pomace andchickpeas were used as the substrate for fibrinolytic enzymeproduction in SSF by Bacillus cereus GD55 [20] and Bacillusamyloliquefaciens [21]

Generally enzyme production in a bioprocess is signifi-cantly influenced by medium components such as nitrogenand carbon sources and physical factors such as inoculumpH temperature and incubation time [22] The cost ofmedium components is one of the critical factors in anindustrial perspective because approximately one-third ofthe production cost of enzyme was attributed to growthmedium cost [23] Hence the utilization of locally availableinexpensive substrates could bring down the production cost[24] Medium optimization is also an important factor inenzyme bioprocess Optimized media enhance the enzymeyield and reduce the production cost Optimization of pro-cess parameters through a one-factor-at-a-time approach isuseful however it fails to provide the interactions betweenthe variables or factors The effective method is statisticaloptimization wherein the medium components having themost significant effect on bioprocess can be identified andoptimized [25]

Central composite rotational design (CCRD) and re-sponse surface methodology (RSM) elucidate the best-yield-ing concentration of each factor in the fermentation processThe number of variables that can be studied with lowernumber of experiments is more in RSM This saves cost andtime and makes RSM more effective than classical methods[26] RSM has been applied to various cases of enzymeproduction [27ndash29] In recent years proteolytic enzymeshave attracted much more attention for their hydrolyticactivity toward recalcitrant animal proteins such as keratin[30] and amyloid prion proteins [31] and collagen [32] andblood clot [33] and management of industrial and medicalwastes Screening of the significant factors was elucidated by

two-level full-factorial design and optimization of the signifi-cant variables using RSM [25] Bacterial fibrinolytic enzymesespecially Bacillus sp can be effectively utilized to cure orprevent cardiovascular diseases [34] In the present studysequential optimization strategy was followed to enhancefibrinolytic enzyme production from Bacillus sp IND12 uti-lizing cow dung as the solid substrate

2 Materials and Methods

21 Screening

Step 1 (screening of protease-producing organisms) Soilfish and rice were used as the sample sources for the produc-tion of fibrinolytic enzyme Soil sample was collected from anagricultural field Rice was boiled for 45min and allowed foraerobic fermentation for a period of 48 h at room temperature(30 plusmn 2∘C) and used as the source of bacteria The marinefish Sardinella longiceps was collected from KanyakumariCoast Tamil Nadu India for the isolation ofmarine bacterialisolates One gram of sample was transferred to an Erlen-meyer flask (100mL) with 50mL of double-distilled water Itwas shaken for 10min and this was resuspended in distilledwater and aliquots were spread on skimmed milk agar plates(skimmed milk 10 beef extract 15 peptic digest of animaltissue 50 yeast extract 15 sodium chloride 50 agar 15bromocresol green 0015 [gL] pH 70) [35] For marineisolates 3 (vw) sodium chloridewas addedwith the culturemediumThe potent proteolytic enzyme-producing bacterialisolate was calculated on the basis of 119875119911 value as described byPrice et al [36] The bacterial isolate with low 119875119911 value wasconsidered for optimization of enzyme production119875119911 value was calculated by using the following equation

119875119911 =Colon diameter

Colon diameter + Zone of precipitation (1)

The isolated protease-producing bacteria were cultured indi-vidually in nutrient broth medium

This was performed by inoculating 1 (vv) preculturedprotease positive isolates and incubated at 37∘C for 48 h Thecell-free supernatant was used as the source of fibrinolyticenzyme

Step 2 (screening of fibrinolytic enzyme-producing bacterialisolates (secondary screening)) Fibrinolytic activity of thecrude enzyme was tested in a fibrin plate composed of 01Msodium phosphate buffer (pH 74) 1 (wv) agarose 12(vv) fibrinogen and thrombin (100 NIH unitsmL) Thefibrin plate was allowed to stand for 1 h at room temperatureto form a fibrin clot layer About 20120583L of crude enzymewas dropped into holes and incubated at 37∘C for 5 h Thefibrinolytic enzyme exhibited a clear zone of degradation offibrin around the well thus indicating its fibrinolytic activity[37] Ten organisms were screened and potent organism wasretained for this study The potent isolate was subjected tobiochemical characterization and 16S rDNA sequencing

Step 3 (fibrin zymography) Fibrin zymography was per-formed to determine fibrinolytic activity of protein having

BioMed Research International 3

different molecular weight Bovine fibrinogen (012) andbovine thrombin (10 NIH UmL) were copolymerized with12 (wv) sodium dodecyl sulfate- (SDS-) polyacrylamidegel After the electrophoresis run the gel was incubated in50mM Tris buffer (pH 74) containing 25 (vv) Triton X-100 and incubated in zymogram reaction buffer (Tris bufferpH 74 and 50mM) for 5 h at 37∘C At the last step the gelwas subjected to staining with Coomassie Brilliant Blue R-250 for a period of 1 h after which it was destained and bandscorresponding to fibrinolytic activities were observed as theunstained regions [38]

Step 4 (in vitro blood clot lytic activity of fibrinolyticenzymes) The clot lytic activity was studied by incubatingthe crude extract with goat blood in vitroThe goat blood clotwas severed into small pieces (400plusmn50mg) and 200U strep-tokinase (positive control) sodium phosphate buffer (nega-tive control) and 200Ufibrinolytic enzymewere addedThenthe mix was incubated at 30 plusmn 2∘C for 24 h and the resultswere observed Based on primary and secondary screeningfibrin zymography and in vitro blood clot lytic activity of thecrude fibrinolytic enzyme the potent organism was selectedfor further studies

22 Identification of Potent Strain The screened bacterialstrain IND12 with highest fibrinolytic enzyme activity wasidentified based on biochemical characters [39] and 16SrRNA gene sequencing [40] The genomic DNA of the strainIND12 was extracted by using a QIAGEN kit (Germany)The16S rRNA gene of the strain was amplified using the upstreamprimer (P1 51015840-AGAGTTTGATCMTGGCTAG-31015840) and thedownstream primer (P2 51015840-ACGGGCGG TGTGTRC-31015840)(Sigma-Aldrich) The amplified product was sequenced andsequence comparison with databases was performed usingBLAST through the NCBI server [41]The isolated bacteriumwas identified as Bacillus sp IND12 The 16S rDNA sequencewas submitted in GenBank database under the accessionnumber KF638633

23 Maintenance of Culture Bacillus sp IND12 was grownon nutrient agar slant which consisted of 05 (wv) pepticdigest of animal tissue 015 (wv) beef extract 015 yeastextract 05 sodium chloride and 15 (wv) agar Thenutrient agar slant was incubated at 37∘C for 24 h and thestrain was stored at minus20∘C as the glycerol stock

24 Inoculum Preparation Seed culture for fibrinolyticenzyme production was prepared by inoculating a loopfulculture of this strain into nutrient broth medium (pepticdigest of animal tissue 50 beef extract 15 yeast extract 15and sodium chloride 50 [gL]) The 250-mL culture flaskwas further incubated at 37∘C for 24 h in a rotary shaker at175 rpm and used as inoculum

25 Screening of Different Agroindustrial Waste Materials forthe Production of Fibrinolytic Enzyme by Solid-State Fermen-tation The substrates such as wheat bran and rice bran werecollected from the local market Green gram husk tapiocapeel and banana peel were processed separately and cow

dung was collected from the farm house All substrateswere dried for seven days powdered and sieved (10ndash15mmin size) 50 g of substrates was taken in Erlenmeyer flaskindividually and Tris buffer (pH 80 01M) was added tomaintain the moisture level (100 vw) The contents ofthe Erlenmeyer flasks were mixed and autoclaved at 15 lbsfor 15min The Erlenmeyer flasks were inoculated with 10inoculum (0810 plusmn 0250OD at 600 nm) and incubated at37∘C for 72 h 50mL double-distilled water was added to thefermented medium It was placed in an orbital shaker for30min at 150 rpm at room temperature (30plusmn2∘C)Themixedslurrywas squeezed through a cheese cloth and centrifuged at4∘C for 20min at 10000 rpm The cell-free clear supernatantwas used as the source of enzyme

26 Fibrinolytic Enzyme Assay Fibrinolytic enzyme wasassayed by the hydrolysis of fibrin [42] The reaction mixturecontained 25 mL of fibrin (12 wv) 25mL of Tris-HCl(01M) containing 001M CaCl2 (pH 78) and a suitableamount of enzymeThe incubationwas carried out for 30min(37∘C) and the measurement time fell within the linearportion of the progress curve The reaction was stopped byadding 5mL trichloroacetic acid (TCA) containing 022Msodium acetate and 033M acetic acid The absorbance ofthe supernatant was measured at 275 nm One fibrinolyticenzyme unit was defined as the amount of sample that gavean increase in absorbency at 275 nm equivalent to 1120583g oftyrosine per min at 37∘C

27 Protease Assay Casein was used as the substrate fordetermining the protease activity The reaction mixture con-tained casein (prepared in 005M of Tris-HCl buffer pH 80)and 01mL of enzyme solution This mixture was incubatedfor 30min at 37∘C and the measurement time fell withinthe linear portion of the progress curve The reaction wasstopped by adding 25mL of TCA (011M) and the mixturewas centrifuged at 10000timesg (10min) The optical density ofthe solution was read against sample blank at 280 nm Oneunit of the protease activity was defined as 1120583g of tyrosineliberated perminute under assay conditionsThe total proteincontent of the crude enzyme was determined as described byLowry et al [43]

28 Screening of Variables for the Production of FibrinolyticEnzyme by One-Variable-at-a-Time Approach The ldquoone-variable-at-a-timerdquo strategy was applied in order to optimizethe various nutrient parameters required for fibrinolyticenzyme production Cow dung (5 g) was used as the substratefor fibrinolytic enzyme production in SSF Each subsequentnutrient factor was examined after taking into account thepreviously optimized factor In the present study themediumcomponents such as carbon sources (1 [ww] sucrosemaltose xylose glucose trehalose and starch) nitrogensources (1 [ww] casein yeast extract urea gelatin pep-tone and beef extract) and inorganic salts (01 [ww] cal-cium chloride ammonium chloride ferrous sulfate ammo-nium sulfate disodium hydrogen phosphate sodium nitratemagnesium chloride and sodium dihydrogen phosphate)were tested The enzyme was extracted as described earlier


and enzyme assay was carried out All experiments wereperformed in triplicate and expressed as mean plusmn standarddeviation Analysis of variance (ANOVA) was carried out tofind the significance of variance

29 Selection of Significant Variables by 25 Full-FactorialDesign Two-level full-factorial design was employed to elu-cidate the relative importance of various medium compo-nents affecting fibrinolytic enzyme production To determinethe significant nutrient and physical factors for fibrinolyticenzymeproduction five variableswere selectedThevariablesused were moisture pH sucrose peptone andMgSO4 at twolevels (ie minus1 and +1) The software Design-Expert (version906) was used to design and analyze the data The meanvalue of ldquominus1 and +1rdquo was used to evaluate the average responseof this model design

119884 = 1205720 +sum120572119894119909119894119894

+sum120572119894119895119909119894119909119895119894119895

+sum120572119894119895119896119909119894119909119895119909119896119894119895119896

+sum120572119894119895119896119897119909119894119909119895119909119896119909119897119894119895119896119897

+sum120572119894119895119896119897119898119909119894119909119895119909119896119909119897119909119898119894119895119896119897119898

(2)

where 119884 is the response 120572119894119895 = 119894119895th 120572119894119895119896 = 119894119895119896th 120572119894119895119896119897 =119894119895119896119897th and 120572119894119895119896119897119898 = 119894119895119896119897119898th interaction coefficients 1205720 wasan intercept and 120572119894 was the 119894th linear coefficient

210 Optimization of Medium Components by RSM CCRDwas employed to determine the best combination for fib-rinolytic enzyme production A total number of 20 sets ofexperimental runs with different combination of physicaland nutrient factors were performed (14 noncentral pointsand 6 central points) Each variable was tested at five levels(minus120572 minus1 0 +1 and +120572) The other factors such as peptonepH and inoculum were set at middle level The fibrinolyticenzyme activity was assayed in triplicate and the averagevalue was reported as response (119884)The results obtained fromthis model were subjected to analysis of variance (ANOVA)for analyzing the regression coefficient The results werebest fitted with the second-order polynomial equation by amultiple regression technique The combination of variablesthat showed themaximum response was determined and thefitted model was validated The suitability of the polynomialmodel equation was judged by determining 1198772 and itssignificance was determined by 119865-test

The second-order polynomial describing a system withthree factors is as follows

119884 = 1205730 +sum3

120573119894119883119894119894=1

+sum3

1205731198941198941198831198942

119894=1

+sum3

120573119894119895119883119894119895119894119895=1

(3)

where 119884 is the response (fibrinolytic activity) 1205730 and 120573119894 arethe offset and coefficients of linear terms respectively and 120573119894119894and 120573119894119895 are the coefficients of square terms and coefficientsof interactive terms respectively 119883119894s were 119860 119861 and 119862 119883119894119895swere 119860119861 119860119862 and 119861119862 (119860-coded value of moisture 119861-codedvalue of sucrose and 119862-coded value of MgSO4)

Design-Expert 906 was the statistical software usedto design and analyze the experimental data The fitted

quadratic model of the experimental trial was expressedin the form of 3D response surface graphs These 3Dgraphs illustrate the main effect and interactive effects of theindependent factors on the dependent factor The predictedconditions were used to perform experiments in order tovalidate the generated model

211 Digestion of Proteins Crude enzyme was appropriatelydiluted at 50UmL concentrations in 50mM Tris-HCl buffer(pH 80) In the present study crude enzyme was incubatedwith goat blood clot (10 g) egg white (10 g) chicken skin(10 g) and bovine serum albumin (1 wv) for 24 h at roomtemperature (30plusmn2∘C)The substrate that was incubated withbuffer was considered as controlThe remaining total proteinwas estimated and the digested protein contentwas calculated()

3 Results and Discussion

Fibrinolytic treatment such as administration of urokinaseintravenously is widely used but the enzyme is expensive andthere is risk of internal hemorrhagewithin the intestinal tractSo research has been pursued to improve the thrombolytictherapy in terms of efficacy and specificity In the presentstudy many potent bacterial isolates were isolated from thesoil sample fishes and cooked rice for the production offibrinolytic enzymes

31 Screening of Bacterial Isolate for Fibrinolytic Enzyme Pro-duction The fibrinolytic enzyme produced by the bacterialisolate was primarily determined by skimmed milk agarplates 119875119911 value was found to be much less for Bacillus spIND12 (020) and this low 119875119911 value indicated good proteaseproduction (Figure 1(a)) Fibrinolytic enzyme is a subclassof proteases and has an ability to degrade fibrin substrate[44] Hence in this study the protease secreting ability of thebacterial isolates was initially screened using skimmed milkagar plates All the selected 10 isolates showed fibrinolyticactivity on this plate (Figure 1(b)) Fibrin plate method is themost suitable method for the determination of fibrinolyticactivity from bacteria fungi and other sources In additionto these fibrin zymography and in vitro blood clot lyticactivity of the crude enzyme of all ten isolates were studiedMost of the selected bacterial isolates synthesized morethan one fibrinolytic enzyme The molecular weight of thefibrinolytic enzyme ranges from 15 to 90 kDa (Figure 1(c))The Bacillus sp such as B subtilis 168 B subtilis CH3ndash5 Bamyloliquefaciens CH51 B amyloliquefaciens CH86 and Blicheniformis CH3ndash17 produced more than one fibrinolyticenzyme at this range [45]The fibrinolytic enzymes graduallydissolved blood clot within 24 h of incubation at roomtemperature (30 plusmn 2∘C) Most of the bacterial fibrinolyticenzymes dissolved blood clot completely (Table 1) Theseresults suggested that fibrinolytic enzymes had obvious effecton dissolving blood clot This kind of clot lysis was reportedwith B subtilis LD-8547 fibrinolytic enzymes [46] Based onthese four stepsrsquo screening procedure IND12 showed potentactivity The selected organism was rod shaped and oxidase-and catalase-positive It hydrolyzed casein and starch and


11 IND12 13 14

15 16 17 18

19 20

(a)

11

C

IND12

1514

13

1920

18

17

16

(b)11

MM

14201

294366974

14201

294366974KDa

KDa15141312 19 20181716

(c)

Figure 1 Screening of bacterial isolates for proteolytic activityThe strain IND12 showed a large halo zone compared to other tested bacterialisolates (a) Screening of fibrinolytic enzyme from the selected bacteria Fibrinolytic activity appeared as a halo zone from the protease positivestrains 11ndash20 (b) Fibrinolytic enzyme activity on SDS-PAGE The strain IND12 showed potent activity (c)

Table 1 In vitro blood clot lytic activity of fibrinolytic enzymes fromthe bacterial isolates

Bacterial strains of blood clot lysis11 10012 (IND12) 10013 87 plusmn 1214 79 plusmn 7115 10016 983 plusmn 14817 637 plusmn 10918 534 plusmn 1219 10020 100

tested negative for gelatin hydrolysis It had tested negative forindole formation citrate utilization andH2S productionTheproteolytic enzymeproduction by any bacterial isolatemainlydepends on the growth media and other physical factorsHowever the optimum concentration of the medium variesfrom strain to strain [47] Hence it is important to screenthe medium components and physical factors for the newbacterial isolates

32 Cow Dung Is a Potential Medium for the Production ofFibrinolytic Enzyme Thebacterial isolateBacillus sp IND12utilized all tested agroindustrial residues for the productionof fibrinolytic enzyme This organism utilized cow dung

Table 2 Effect of agroresidues on fibrinolytic enzyme productionin SSF

Agroresidues Fibrinolytic activity (Ug)Banana peel 85Cow dung 687Green gram husk 493Rice bran 273Tapioca peel 384Wheat bran 640

effectively for its growth and fibrinolytic enzyme productionEnzyme production was high in the cow dung substrate(687 plusmn 65Ug) (Table 2) The selection of an ideal substratefor enzyme production in SSF depends on several factorsincluding availability and cost [9] and nutritive value of themedium [29] Cow dung is one of the unexploited and mostabundant feedstock for fibrinolytic enzyme production Itcontains cellulose (354ndash376) hemicelluloses (326ndash341)ash (133ndash134) nitrogen (12ndash14) and other essentialnutrients such as Mg Ca Zn S Cu B and Mn [48 49] Thecarbon nitrogen and other nutrientsrsquo content in cow dungare an indication that it could be a good nutrient source forthe microbes [50] The agroindustrial residues such as ricechaff [51] shrimp shells [4] cane molasses [52] and wheatbran [7] were used as the substrate for the production offibrinolytic enzyme The availability of cow dung substrateis higher than most of the reported substrates Considering


the availability and cost cow dung is an ideal medium for theproduction of fibrinolytic enzyme from Bacillus sp IND12The growth of this strain on cow dung is important for theproduction of various industrially useful enzymes in cheapcost

33 Effect of Carbon Nitrogen and Ionic Sources To screendifferent carbon sources 1 carbon source (maltose glucosesucrose xylose starch and trehalose) was supplementedwith cow dung substrate This medium was inoculated with10 inoculum (0810 plusmn 0250OD at 600 nm) and incubatedfor 72 h All tested carbon sources enhanced fibrinolyticenzyme production (Figure 2) The influence of differentcarbon sources on fibrinolytic enzyme production was foundto be statistically significant (119875 lt 000001) by the one-way ANOVA The lowest fibrinolytic enzyme activity wasobtained with xylose (648 plusmn 115Ug) and highest enzymeactivity was observedwith sucrose (1049plusmn1743Ug) Similarresults were obtained in the study of Liu et al [25] in thescreening of the carbon sources on the fibrinolytic activityof Bacillus natto NLSSE Considering the cheap cost andenzyme production sucrose was chosen as the carbon sourcefor further optimization study To screen different nitrogensources 1 casein peptone beef extract yeast extract ureaand gelatin were supplemented with cow dung substrate Thelowest fibrinolytic activity was registered with urea (435 plusmn387Ug) and the highest with peptone (1127 plusmn 138Ug)(Figure 2) The influence of different nitrogen sources onfibrinolytic enzyme production was found to be statisticallysignificant (119875 lt 000001) by the one-way ANOVA Similarresults were obtained with Bacillus subtilis RJAS19 [53] andStreptomyces sp [54] From this result peptone was chosenas the nitrogen source for further optimization study Theculturemediumwas supplementedwith 01 ions such as cal-cium chloride ammonium chloride ferrous sulfate ammo-nium sulfate disodium hydrogen phosphate sodium nitrateand sodium dihydrogen phosphate Enzyme production wasfound to be high in the medium containing MgSO4 as theionic source The ions such as Ca2+ and Mn2+ also enhancedthe production of fibrinolytic enzyme and enzyme activitywas 8674plusmn49 and 9126plusmn38Ug respectively (Figure 2)Theinfluence of different ionic sources on fibrinolytic enzymeproduction was found to be statistically significant (119875 lt000001) by the one-wayANOVA In this study Ca2+ ions alsopositively regulated enzyme productionThe positive effect ofdivalent ions such as Mg2+ and Mn2+ was reported by Pillaiet al [55] with Bacillus subtilis P13 The stimulating effect ofCaCl2 was also stated by Mabrouk et al [56] The presenceof other components at 01 level did not positively influencethe enzyme production

34 Studies on the Effect of Process Variables on FibrinolyticEnzyme Production by 25 Full-Factorial Design The signif-icance of five medium components namely moisture (119860)pH (119861) sucrose (119862) peptone (119863) and MgSO4 (119864) for theproduction of fibrinolytic enzyme was elucidated as givenby 25 full-factorial design The factors and their levels weredescribed in detail (Table 3) The 25 full-factorial experiment

0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Star

ch

Xylo

se

Sucr

ose

Con

trol

Mal

tose

Glu

cose

Treh

alos

e

Carbon sources ()

0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Beef

extr

act

Gel

atin

Con

trol

Yeas

t ext

ract

Case

in

Ure

a

Pept

one

Nitrogen sources ()

0100200300400500600700800900

1000

Enzy

me a

ctiv

ity (U

g)

MnC

l 2

Na 2

HPO

4

Na 2

HPO

4

Con

trol

Am

chl

orid

e

Ferr

ous s

ulfa

te

Sodi

um n

itrat

e

Calc

ium

chlo

ride

Ions ()Figure 2 Effect of carbon sources nitrogen sources and ions onfibrinolytic enzymeproduction byBacillus sp IND12These nutrientsources were supplemented individually with cow dung substrateinoculated with 10 inoculum (0810 plusmn 0250OD at 600 nm) andincubated at 37∘C for 72 h

Table 3 Variables and their levels for 25 full-factorial design

Symbol Variable name Units Coded levelsminus1 +1

119860 Moisture 90 120119861 pH 7 9119862 Sucrose 01 1119863 Peptone 01 1119864 MgSO4 001 01


Table 4 Two-level full-factorial design for selection of significant process variables for fibrinolytic enzyme production from Bacillus spIND12

Run Moisture(A)

pH(B)

Sucrose(C)

Peptone(D)

MgSO4(E) Enzyme activity (Ug)

1 1 1 1 1 1 19202 minus1 1 1 1 1 14193 1 minus1 1 1 1 39494 minus1 minus1 1 1 1 31225 1 1 minus1 1 1 14736 minus1 1 minus1 1 1 19497 1 minus1 minus1 1 1 11668 minus1 minus1 minus1 1 1 15219 1 1 1 minus1 1 318410 minus1 1 1 minus1 1 146611 1 minus1 1 minus1 1 103212 minus1 minus1 1 minus1 1 106613 1 1 minus1 minus1 1 170214 minus1 1 minus1 minus1 1 388115 1 minus1 minus1 minus1 1 159016 minus1 minus1 minus1 minus1 1 29217 1 1 1 1 minus1 303818 minus1 1 1 1 minus1 137719 1 minus1 1 1 minus1 164220 minus1 minus1 1 1 minus1 149021 1 1 minus1 1 minus1 163822 minus1 1 minus1 1 minus1 93323 1 minus1 minus1 1 minus1 110024 minus1 minus1 minus1 1 minus1 100225 1 1 1 minus1 minus1 82326 minus1 1 1 minus1 minus1 43927 1 minus1 1 minus1 minus1 253028 minus1 minus1 1 minus1 minus1 140829 1 1 minus1 minus1 minus1 150930 minus1 1 minus1 minus1 minus1 113931 1 minus1 minus1 minus1 minus1 106232 minus1 minus1 minus1 minus1 minus1 1094

showed wide variation of enzyme activity In the presentstudy all factors positively regulated fibrinolytic enzymeproduction The fibrinolytic enzyme production by Bacillussp IND12 varied from 292 to 3949Ug in 32 experimentswhich suggests the significance of medium components andtheir concentration on fibrinolytic enzyme production Theresults showed that run number 3 resulted in the high-est fibrinolytic enzyme production (3949Ug) followed bymedium composition in run 14 (3881Ug) (Table 4) Theobservedmean response of this two-level full-factorial designwas 162113Ug The main effect 119865 value and 119875 value ofeach factor are given in Table 5 According to these ANOVAresults the five variables such as 119860 119861 119862 119863 and 119864 werestatistically significant on fibrinolytic enzyme productionAmong the variables the most significant variable was

moisture which had highest correlation coefficient com-pared to the other tested factors The linear interactive andquadratic coefficients such as 119860119862 119860119864 119861119862 119861119864 119862119863 119860119861119862119860119861119863 119860119861119864 119860119862119864 119861119862119864 119861119863119864 119862119863119864 119860119861119862119863 119860119861119862119864 119861119862119863119864and119860119861119862119863119864 were also statistically significantThe regressionequation involving the coded factors is

Enzyme activity (119884) = +165488 + 180119860 + 8825119861

+ 21419119862 + 14131119863

+ 26588119864 + 21569119860119862

minus 9875119860119864 minus 24906119861119862

minus 16606119861119863 + 11525119861119864


Table 5 Results of ANOVA for the two-level full-factorial design

Source Sum of squares df Mean square F value 119875 valueModel 250119864 + 07 22 113119864 + 06 8509 lt00001A-moisture 104119864 + 06 1 104119864 + 06 7779 lt00001B-pH 249119864 + 05 1 249119864 + 05 187 00019C-sucrose 147119864 + 06 1 147119864 + 06 11015 lt00001D-peptone 639119864 + 05 1 639119864 + 05 4795 lt00001E-MgSO4 226119864 + 06 1 226119864 + 06 16973 lt00001AC 149119864 + 06 1 149119864 + 06 1117 lt00001AE 312119864 + 05 1 312119864 + 05 2341 00009BC 199119864 + 06 1 199119864 + 06 14894 lt00001BD 883119864 + 05 1 883119864 + 05 6621 lt00001BE 425119864 + 05 1 425119864 + 05 3189 00003CD 176119864 + 06 1 176119864 + 06 13175 lt00001ABC 716119864 + 05 1 716119864 + 05 5371 lt00001ABD 435119864 + 05 1 435119864 + 05 3262 00003ABE 488119864 + 05 1 488119864 + 05 3662 00002ACE 203119864 + 05 1 203119864 + 05 152 00036BCE 333119864 + 05 1 333119864 + 05 2495 000017BDE 543119864 + 06 1 543119864 + 06 40768 lt00001CDE 202119864 + 05 1 202119864 + 05 1513 00037ABCD 360119864 + 05 1 360119864 + 05 2698 00006ABCE 865119864 + 05 1 865119864 + 05 6492 lt00001BCDE 1665119864 + 0006 1 1665119864 + 0006 12495 lt00001ABCDE 175119864 + 06 1 175119864 + 06 13105 lt00001Residual 120119864 + 05 9 1332768Cor total 251119864 + 07 31

Table 6 Variables and their levels for central composite rotational design

Variables Symbol Coded valuesminus120572 minus1 0 +1 +120572

Moisture () 119860 8318 90 100 110 11682Sucrose () 119861 033 05 075 10 117MgSO4 () 119862 003 005 008 01 012

+ 23425119862119863 + 14956119860119861119862

+ 11656119860119861119863 minus 1235119860119861119864

+ 7956119860119862119864 minus 10194119861119862119864

minus 41206119861119863119864 + 7937119862119863119864

minus 106119860119861119862119863 + 16444119860119861119862119864

minus 22813119861119862119863119864

minus 23362119860119861119862119863119864(4)

Among the factors the coefficient estimate was high formoisture sucrose and MgSO4 Hence these three factorswere selected for further studies The middle value of theother two factors (05 (ww) peptone and pH 80) was usedfor CCRD

35 Response Surface Methodology A CCRD was employedto find the interactions among significant independent fac-tors (moisture sucrose and MgSO4) Each variable was ana-lyzed at five coded levels (minus120572 minus1 0 +1 +120572) (Table 6) Enzymeassaywas carried out and the experimental result was taken asresponse119884 (Table 7)The119865-test for anANOVAwas generatedto elucidate the significance of the model and understandthe reliability of the regression model The selected variablesmoisture sucrose and MgSO4 were found to be significant(119875 lt 005) (Table 8) The interactive effects such as 119860119861119861119862 1198602 1198612 and 1198622 were also statistically significant Thedeterminant coefficient termed1198772 was calculated to be 09933for fibrinolytic enzyme production by Bacillus sp IND12suggesting 9933 of the variability in the response The finalequation in terms of coded factor can be written as follows

Fibrinolytic enzyme (119884)

= +292144 + 34022119860 minus 14718119861 minus 54364119862


Table 7 The matrix of the CCRD experiment and fibrinolytic enzyme activity

Run Moisture(119860)

Sucrose(119861)

MgSO4(119862) Enzyme activity (Ug)

1 minus1 1 1 11982 0 0 0 30473 0 0 0 27404 1 1 minus1 35085 0 0 0 28906 0 minus1682 0 15097 1 minus1 1 35058 0 0 1682 39729 0 0 0 275410 minus1 minus1 1 214211 0 0 minus1682 591612 1 minus1 minus1 404813 1682 0 0 303214 minus1682 0 0 186115 0 1682 0 104716 1 1 1 167017 minus1 minus1 minus1 231418 0 0 0 294019 minus1 1 minus1 360020 0 0 0 3150

Table 8 Results of ANOVA for the CCRD

Source Sum of squares df Mean square F value 119875 valueModel 2247119864 + 007 9 2497119864 + 006 16597 lt00001119860-moisture 1581119864 + 006 1 1581119864 + 006 10507 lt00001119861-sucrose 2958119864 + 005 1 2958119864 + 005 1966 00013119862-MgSO4 4036119864 + 006 1 4036119864 + 006 26826 lt00001119860119861 1546119864 + 006 1 1546119864 + 006 10276 lt00001119860119862 4605613 1 4605613 306 01108119861119862 9282119864 + 005 1 9282119864 + 005 6169 lt000011198602 4454119864 + 005 1 4454119864 + 005 2960 000031198612 4998119864 + 006 1 4998119864 + 006 33221 lt000011198622 7207119864 + 006 1 7207119864 + 006 47903 lt00001Residual 1505119864 + 005 10Lack of fit 2017351 5 4034702 298 0131Pure error 1303119864 + 005 5Cor total 2263119864 + 007 19

minus 43962119860119861 minus 7587119860119862 minus 34062119861119862 minus 175811198602

minus 588931198612 + 707191198622(5)

where119884was the response of fibrinolytic yield and119860 119861 and119862were the coded terms for independent variables of moisturesucrose and MgSO4 respectively The interactions of vari-ables in the model were demonstrated as 3D response surfaceplots (Figures 3(a)ndash3(c))The1198772 value of the predictedmodelwas 09848 whichwas reasonable in agreement with adjusted

1198772 of 09874 The adequate precision reflects on the signal-to-noise ratio the ratio of 55662 indicates adequate signalof the model The response surface curve shows the inter-action between moisture and sucrose Enzyme productionwas maximum at higher moisture content and decreased atlower moisture content (Figure 3(a)) The moisture contentof SSF medium takes different optimum values with differingsubstrates It was reported that 40 was optimum for theproduction of proteolytic enzyme using wheat bran and lentilhusk as the substrate [57] however 140 was registered asthe optimummoisture content for green gram husk substrate


1000

100090

080070

060050 9000

950010000

11000

A moisture ()B sucrose ()

10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)

Figure 3 Response surface curve showing the effect of moisture and sucrose (a) moisture and MgSO4 (b) and sucrose and MgSO4 (c) onthe fibrinolytic activity of Bacillus sp IND12 The other factors (pH and peptone) were kept at middle level

for protease production [47] In earlier reports of fibrinolyticenzyme production many organic carbon sources have beenused in the medium including sucrose for Paenibacillussp IND8 [58] whereas glucose and soybean flour wereused with Streptomyces sp [59] The data suggest that cowdung is a promising substrate for use in fibrinolytic enzymeproduction

The three-dimensional response surface for the interac-tion of MgSO4 and moisture is represented (Figure 3(b))The fibrinolytic enzyme yield was found to be increasedwith higher concentration of moisture The results showedthat fibrinolytic enzyme production was most affected bymoisture levels compared to nutrient factors In SSFmoisturecontent of themedium is critically importantThe interactionof sucrose and MgSO4 indicated that fibrinolytic enzyme

yield was significantly affected by interaction between thesetwo factors (Figure 3(c)) The optimized medium (10973moisture 057 sucrose and 0093MgSO4) showed 4143plusmn1231Ug material which was more than fourfold theunoptimized medium (978 plusmn 364Ug) Deepak et al [60]reported similar optimizationwith RSMwithBacillus subtiliswhich resulted in a twofold increase in fibrinolytic enzymeproduction RSM was also employed for Bacillus sp strainAS-S20-I [61] and Paenibacillus sp IND8 [58] and 4-fold and45-fold enzyme production was reported

36 Validation of the Predicted Model Validation experi-ments to test the appropriateness of the model were con-ducted with the strain IND12 under the following condi-tions 10973 moisture 057 sucrose and 0093 MgSO4


Fibrinolytic enzyme production reached the maximum of4143 plusmn 1231Ug after 72 h incubation which was highlycomparablewith the predicted value (416868plusmn364Ug)Theobserved and predicted values went hand in hand indicatingthe model validation

37 Digestion of Proteins from Natural Sources by Bacillussp IND12 Protease The ability of crude protease to digestsome natural proteins was tested The enzyme digested goatblood clot completely (100 plusmn 092 solubility) within 24 h ofincubation at room temperature (30∘C plusmn 2∘C) These resultsshowed that the fibrinolytic enzyme of Bacillus sp IND12 canconvert the insoluble forms of goat blood clot into solubleform It also hydrolyzed 29 plusmn 49 bovine serum albuminwithin 12 h of incubation This enzyme digested coagulatedegg white (100 plusmn 062 solubility) and also hydrolyzedchicken skin (83 plusmn 36 solubility) The study suggests theusefulness of this enzyme for various applications includingcollagen replacement therapy and waste treatment Thesekinds of proteolytic activity were registered previously withPseudomonas aeruginosa PD100 [62] and Bacillus RVB290[63] The activity of the protease on egg protein chickenskin and blood clot indicates its importance in industrialapplication waste treatment and medicine

4 Conclusion

To conclude four-step screening was successful to evaluatethe potent fibrinolytic enzyme-producing bacterial isolateCow dung was used as the cheap substrate for the productionof fibrinolytic enzyme from Bacillus sp IND12 The opti-mum process parameters were as follows 10973 moisture057 sucrose and 0093 MgSO4 At these conditionsfibrinolytic enzyme production reached the maximum of4143 plusmn 1231Ug after 72 h incubation which was highlycomparable with the predicted value (416868 plusmn 364Ug)This enzyme hydrolyzed goat blood clot chicken skinegg white and bovine serum albumin which suggestedits applications in clinical and waste water treatment Thebioprocessing of this low cost substrate can minimize theproduction cost of enzymeThe enhanced yield of fibrinolyticenzyme by Bacillus sp IND12 using cow dung substrate couldbe useful for the production of enzyme in an industrialscale

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Ponnuswamy Vijayaraghavan and Arumugaperumal Arunperformed the majority of the experiments Naif AbdullahAl-Dhabi Mariadhas Valan Arasu Oh Young Kwon andYoungOckKim analyzed the experimental data P Rajendranwas involved in sequential screening assays Samuel GnanaPrakash Vincent was involved in designing part of the exper-iments All authors read and approved the final manuscript

Acknowledgments

The funding from the Deanship of Scientific Research at KingSaud University (PRG-1437-28) was sincerely appreciated

References

[1] NMackman ldquoTriggers targets and treatments for thrombosisrdquoNature vol 451 no 7181 pp 914ndash918 2008

[2] K I Fayek and S T ElminusSayed ldquoSome properties of two purifiedfibrinolytic enzymes from Bacillus subtilis and B polymyxardquoZeitschrift fur Allgemeine Mikrobiologie vol 20 no 6 pp 383ndash387 1980

[3] H Malke and J J Ferretti ldquoStreptokinase cloning expressionand excretion by Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 81 no11 pp 3557ndash3561 1984

[4] S-LWang H-J Chen T-W Liang and Y-D Lin ldquoA novel nat-tokinase produced by Pseudomonas sp TKU015 using shrimpshells as substraterdquo Process Biochemistry vol 44 no 1 pp 70ndash76 2009

[5] Y Uesugi H Usuki M Iwabuchi and T Hatanaka ldquoHighlypotent fibrinolytic serine protease from Streptomycesrdquo Enzymeand Microbial Technology vol 48 pp 7ndash12 2011

[6] P Vijayaraghavan S G P Vincent and M Valan ArasuldquoPurification characterization of a novel fibrinolytic enzymefrom Paenibacillus sp IND8 and its in vitro thrombolyticactivityrdquo South Indian Journal of Biological Sciences vol 2 no4 pp 434ndash444 2016

[7] P Vijayaraghavan and S G Prakash Vincent ldquoStatistical opti-mization of fibrinolytic enzyme production using agroresiduesby Bacillus cereus IND1 and its thrombolytic activity in vitrordquoBioMed Research International vol 2014 Article ID 725064 11pages 2014

[8] C-T Chang P-MWang Y-F Hung and Y-C Chung ldquoPurifi-cation and biochemical properties of a fibrinolytic enzyme fromBacillus subtilis-fermented red beanrdquo Food Chemistry vol 133no 4 pp 1611ndash1617 2012

[9] A Pandey C R Soccol P Nigam D Brand R Mohan and SRoussos ldquoBiotechnological potential of coffee pulp and coffeehusk for bioprocessesrdquo Biochemical Engineering Journal vol 6no 2 pp 153ndash162 2000

[10] B Johnvesly B R Manjunath and G R Naik ldquoPigeon peawaste as a novel inexpensive substrate for production of a ther-mostable alkaline protease from thermoalkalophilic Bacillus spJB-99rdquo Bioresource Technology vol 82 no 1 pp 61ndash64 2002

[11] A K Mukherjee H Adhikari and S K Rai ldquoProduction ofalkaline protease by a thermophilic Bacillus subtilis under solid-state fermentation (SSF) condition using Imperata cylindricagrass and potato peel as low-cost medium characterization andapplication of enzyme in detergent formulationrdquo BiochemicalEngineering Journal vol 39 no 2 pp 353ndash361 2008

[12] G D Saratale S D Kshirsagar V T Sampange et al ldquoCel-lulolytic enzymes production by utilizing agricultural wastesunder solid state fermentation and its application for biohydro-gen productionrdquo Applied Biochemistry and Biotechnology vol174 no 8 pp 2801ndash2817 2014

[13] T Paul A Das A Mandal et al ldquoEffective dehairing propertiesof keratinase from Paenibacillus woosongensis TKB2 obtainedunder solid state fermentationrdquo Waste and Biomass Valoriza-tion vol 5 no 1 pp 97ndash107 2014


[14] B L Maiorella and F J Castillo ldquoEthanol biomass and enzymeproduction for whey waste abatementrdquo Process Biochemistryvol 19 no 4 pp 157ndash161 1984

[15] E Ravindranath K Chitra S Shamshath Begum and AN Gopalakrishnan ldquoEffect of recirculation rate on anaerobictreatment of fleshing using UASB reactor with recovery ofenergyrdquo Journal of Scientific and Industrial Research vol 69 pp90ndash793 2010

[16] R Siala F Frikha S Mhamdi M Nasri and A S KamounldquoOptimization of acid protease production by Aspergillus nigerI1 on shrimp peptone using statistical experimental designrdquoTheScientific World Journal vol 2012 Article ID 564932 11 pages2012

[17] A E Asagbra A I Sanni and O B Oyewole ldquoSolid-state fer-mentation production of tetracycline by Streptomyces strainsusing some agricultural wastes as substraterdquo World Journal ofMicrobiology and Biotechnology vol 21 no 2 pp 107ndash114 2005

[18] K Prasad Kota and P Sridhar ldquoSolid state cultivation of Strep-tomyces clavuligerus for cephamycin C productionrdquo ProcessBiochemistry vol 34 no 4 pp 325ndash328 1999

[19] D Chakradhar S Javeed and A P Sattur ldquoStudies on theproduction of nigerloxin using agro-industrial residues bysolid-state fermentationrdquo Journal of Industrial Microbiology andBiotechnology vol 36 no 9 pp 1179ndash1187 2009

[20] E Venkata Naga Raju and G Divakar ldquoOptimization andproduction of fibrinolytic protease (GD kinase) from differentagro industrial wastes in solid state fermentationrdquo CurrentTrends in Biotechnology and Pharmacy vol 7 no 3 pp 763ndash7722013

[21] X Wei M Luo L Xu et al ldquoProduction of fibrinolytic enzymefrom Bacillus amyloliquefaciens by fermentation of chickpeaswith the evaluation of the anticoagulant and antioxidant prop-erties of chickpeasrdquo Journal of Agricultural and Food Chemistryvol 59 no 8 pp 3957ndash3963 2011

[22] P N Nehete V D Shah and R M Kothari ldquoProfiles of alkalineprotease production as a function of composition of the slantage transfer and isolate number and physiological state ofculturerdquo Biotechnology Letters vol 7 no 6 pp 413ndash418 1985

[23] O Kirk T V Borchert and C C Fuglsang ldquoIndustrial enzymeapplicationsrdquo Current Opinion in Biotechnology vol 13 no 4pp 345ndash351 2002

[24] A Gessesse and B A Gashe ldquoProduction of alkaline proteaseby an alkaliphilic bacteria isolated from an alkaline soda lakerdquoBiotechnology Letters vol 19 no 5 pp 479ndash481 1997

[25] J Liu J Xing T Chang Z Ma and H Liu ldquoOptimization ofnutritional conditions for nattokinase production by Bacillusnatto NLSSE using statistical experimental methodsrdquo ProcessBiochemistry vol 40 no 8 pp 2757ndash2762 2005

[26] D C Montogomery Design and Analysis of Experiments JohnWiley amp Sons New York NY USA 5th edition 2001

[27] H Zhang Q Sang and W Zhang ldquoStatistical optimization ofchitosanase production by Aspergillus sp QD-2 in submergedfermentationrdquoAnnals ofMicrobiology vol 62 no 1 pp 193ndash2012012

[28] B Bhunia and A Dey ldquoStatistical approach for optimization ofphysiochemical requirements on alkaline protease productionfrom Bacillus licheniformis NCIM 2042rdquo Enzyme Research vol2012 Article ID 905804 13 pages 2012

[29] P Vijayaraghavan S G Prakash Vincent and G S DhillonldquoSolid-substrate bioprocessing of cow dung for the productionof carboxymethyl cellulase by Bacillus halodurans IND18rdquoWaste Management vol 48 pp 513ndash520 2016

[30] P Bressollier F LetourneauMUrdaci and B Verneuil ldquoPurifi-cation and characterization of a keratinolytic serine proteinasefrom Streptomyces albidoflavusrdquo Applied and EnvironmentalMicrobiology vol 65 no 6 pp 2570ndash2576 1999

[31] Z Hui H Doi H Kanouchi et al ldquoAlkaline serine proteaseproduced by Streptomyces sp degrades PrP(Sc)rdquo Biochemicaland Biophysical Research Communications vol 321 no 1 pp45ndash50 2004

[32] Y Uesugi J Arima H Usuki M Iwabuchi and T HatanakaldquoTwo bacterial collagenolytic serine proteases have differenttopological specificitiesrdquo Biochimica et Biophysica Acta (BBA)mdashProteins and Proteomics vol 1784 no 4 pp 716ndash726 2008

[33] S Sugimoto T Fujii T Morimiya O Johdo and T NakamuraldquoThe fibrinolytic activity of a novel protease derived froma tempeh producing fungus Fusarium sp BLBrdquo BioscienceBiotechnology and Biochemistry vol 71 no 9 pp 2184ndash21892007

[34] Johnson Yanti M T Suhartono and B W Lay ldquoIsolationand purification of a fibrinolytic enzyme from bacterial strainisolated from tuak an indonesian palm winerdquo InternationalJournal of Pharma andBio Sciences vol 6 no 4 pp B988ndashB9962015

[35] P Vijayaraghavan and S G P Vincent ldquoA simplemethod for thedetection of protease activity on agar plates using bromocresol-green dyerdquo Journal of Biochemical Technology vol 4 no 3 pp628ndash630 2013

[36] M F Price I D Wilkinson and L O Gentry ldquoPlate methodfor detection of phospholipase activity in Candida albicansrdquoSabouraudia vol 20 no 1 pp 7ndash14 1982

[37] T Astrup and S Mullertz ldquoThe fibrin plate method for estimat-ing fibrinolytic activityrdquoArchives of Biochemistry andBiophysicsvol 40 no 2 pp 346ndash351 1952

[38] G M Kim A R Lee K W Lee et al ldquoCharacterization of a 27kDa fibrinolytic enzyme from Bacillus amyloliquefaciensCH51isolated from Cheonggukjangrdquo Journal of Microbiology andBiotechnology vol 19 no 10 pp 997ndash1004 2009

[39] J G Hol N R Krieg P H Sneath J J Stanley and S TWilliams Bergeyrsquos Manual of Determinative Bacteriology Lip-pincott Williams ampWilkins Baltimore Md USA 1994

[40] T S Rejiniemon R R Hussain and B Rajamani ldquoIn-vitrofunctional properties of Lactobacillus plantarum isolated fromfermented ragimaltrdquo South Indian Journal of Biological Sciencesvol 1 no 1 pp 15ndash23 2015

[41] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[42] M L Ansen ldquoThe estimation of pepsin trypsin papain andcathepsinwith haemoglobinrdquo Journal of General Physiology vol22 pp 78ndash79 1939

[43] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[44] M Fujita K Nomura K Hong Y Ito A Asada and SNishimuro ldquoPurification and characterization of a strong fib-rinolytic enzyme (nattokinase) in the vegetable cheese nattoa popular soybean fermented food in Japanrdquo Biochemical andBiophysical Research Communications vol 197 no 3 pp 1340ndash1347 1993

[45] G M Kim A R Lee K W Lee et al ldquoCharacterization ofa 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens


CH51 isolated from Cheonggukjangrdquo Journal of Microbiologyand Biotechnology vol 19 no 2 pp 997ndash1004 2009

[46] J Yuan J Yang Z Zhuang Y Yang L Lin and S WangldquoThrombolytic effects of Douchi Fibrinolytic enzyme fromBacillus subtilis LD-8547 in vitro and in vivordquo BMC Biotechnol-ogy vol 12 article no 36 2012

[47] R S Prakasham C Subba Rao and P N Sarma ldquoGreen gramhusk an inexpensive substrate for alkaline protease productionby Bacillus sp in solid-state fermentationrdquo Bioresource Technol-ogy vol 97 no 13 pp 1449ndash1454 2006

[48] R V Misra R N Roy and H Hiraok On Farm CompostingMethod FAO Rome Italy 2003

[49] C D Fulhage Reduce Environmental Problems with ProperLand Application of Animal Manure University of MissouriExtension New York NY USA 2000

[50] D V Adegunloye F C Adetuyi F A Akinyosoye and MO Doyeni ldquoMicrobial analysis of compost using cowdung asboosterrdquo Pakistan Journal of Nutrition vol 6 no 5 pp 506ndash5102007

[51] S Tao L Peng L Beihui L Deming and L Zuohu ldquoSolid statefermentation of rice chaff for fibrinolytic enzyme production byFusarium oxysporumrdquo Biotechnology Letters vol 19 no 5 pp465ndash467 1997

[52] W Zeng W Li L Shu J Yi G Chen and Z Liang ldquoNon-sterilized fermentative co-production of poly(120574-glutamic acid)and fibrinolytic enzyme by a thermophilic Bacillus subtilisGXA-28rdquo Bioresource Technology vol 142 pp 697ndash700 2013

[53] D J Mukesh Kumar R Rakshitha M Annu Vidhya et alldquoProduction optimization and characterization of fibrinolyticenzyme by Bacillus subtilis RJAS19rdquo Pakistan Journal of Biologi-cal Sciences vol 17 no 4 pp 529ndash534 2014

[54] R R Chitte and S Dey ldquoProduction of a fibrinolytic enzyme bythermophilic Streptomyces speciesrdquoWorld Journal of Microbiol-ogy and Biotechnology vol 18 no 4 pp 289ndash294 2002

[55] P Pillai S Mandge and G Archana ldquoStatistical optimizationof production and tannery applications of a keratinolytic serineprotease fromBacillus subtilisP13rdquoProcess Biochemistry vol 46no 5 pp 1110ndash1117 2011

[56] S S Mabrouk A M Hashem N M A El-Shayeb A-M SIsmail and A F Abdel-Fattah ldquoOptimization of alkaline pro-tease productivity by Bacillus licheniformis ATCC 21415rdquo Biore-source Technology vol 69 no 2 pp 155ndash159 1999

[57] F Uyar and Z Baysal ldquoProduction and optimization of processparameters for alkaline protease production by a newly isolatedBacillus sp under solid state fermentationrdquo Process Biochem-istry vol 39 no 12 pp 1893ndash1898 2004

[58] P Vijayaraghavan and S G P Vincent ldquoMedium optimizationfor the production of fibrinolytic enzyme by Paenibacillussp IND8 using response surface methodologyrdquo The ScientificWorld Journal vol 2014 Article ID 276942 9 pages 2014

[59] GMM Silva R P Bezerra J A Teixeira T S Porto J L Lima-Filho and A L F Porto ldquoFibrinolytic protease productionby new Streptomyces sp DPUA 1576 from Amazon lichensrdquoElectronic Journal of Biotechnology vol 18 no 1 pp 16ndash19 2015

[60] V Deepak K Kalishwaralal S Ramkumarpandian S V BabuS R Senthilkumar and G Sangiliyandi ldquoOptimization ofmedia composition for Nattokinase production by Bacillussubtilis using response surface methodologyrdquo Bioresource Tech-nology vol 99 no 17 pp 8170ndash8174 2008

[61] A K Mukherjee and S K Rai ldquoA statistical approach for theenhanced production of alkaline protease showing fibrinolytic

activity from a newly isolated Gram-negative Bacillus sp strainAS-S20-Irdquo New Biotechnology vol 28 no 2 pp 182ndash189 2011

[62] M F Najafi D Deobagkar and D Deobagkar ldquoPotentialapplication of protease isolated from Pseudomonas aeruginosaPD100rdquo Electronic Journal of Biotechnology vol 8 no 2 pp 197ndash203 2005

[63] S Vijayalakshmi S Venkatkumar andVThankamani ldquoScreen-ing of alkalophilic thermophilic protease isolated from BacillusRVB290 for Industrial applicationrdquo Research in Biotechnologyvol 2 pp 32ndash41 2011

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


both solid-state fermentation (SSF) and submerged fermen-tation processes

SSF has emerged as a useful method for production ofenzymes and many significant pharmaceutical products Ina SSF process usage of convenient substrate is one of theimportant factors and the yield is considerably higher thansubmerged fermentation [9] Agroindustrial residues likewheat bran [9] pigeon pea [10] potato peel [11] grass powder[12] and chicken feather [13] were used for the production ofenzymes Apart from these agroindustrial wastes researchersattempted to use various effluents including dairy and tan-nery industrial effluents for the production of proteolyticenzymes These industrial wastes are rich in nitrogen andcarbon sources Several studies have proved the potentialuse of these industrial wastes to minimize pollution and toincrease the product yield [14 15] The marine food wasteshrimp peptone was also shown to be a potential substratefor SSF involving the production of proteases recently [16]The agricultural wastes such as peanut shell corncob pomaceof corn and peels of cassava were used for the productionof tetracycline [17] The antibiotic Cephamycin C wasproduced in SSF using wheat straw deoiled cake of cottonseed sunflower cake and corn steep liquor [18] and wheatbran and sweet lemon peel was used as the substrate forthe production of nigerloxin in SSF [19] Apple pomace andchickpeas were used as the substrate for fibrinolytic enzymeproduction in SSF by Bacillus cereus GD55 [20] and Bacillusamyloliquefaciens [21]

Generally enzyme production in a bioprocess is signifi-cantly influenced by medium components such as nitrogenand carbon sources and physical factors such as inoculumpH temperature and incubation time [22] The cost ofmedium components is one of the critical factors in anindustrial perspective because approximately one-third ofthe production cost of enzyme was attributed to growthmedium cost [23] Hence the utilization of locally availableinexpensive substrates could bring down the production cost[24] Medium optimization is also an important factor inenzyme bioprocess Optimized media enhance the enzymeyield and reduce the production cost Optimization of pro-cess parameters through a one-factor-at-a-time approach isuseful however it fails to provide the interactions betweenthe variables or factors The effective method is statisticaloptimization wherein the medium components having themost significant effect on bioprocess can be identified andoptimized [25]

Central composite rotational design (CCRD) and re-sponse surface methodology (RSM) elucidate the best-yield-ing concentration of each factor in the fermentation processThe number of variables that can be studied with lowernumber of experiments is more in RSM This saves cost andtime and makes RSM more effective than classical methods[26] RSM has been applied to various cases of enzymeproduction [27ndash29] In recent years proteolytic enzymeshave attracted much more attention for their hydrolyticactivity toward recalcitrant animal proteins such as keratin[30] and amyloid prion proteins [31] and collagen [32] andblood clot [33] and management of industrial and medicalwastes Screening of the significant factors was elucidated by

two-level full-factorial design and optimization of the signifi-cant variables using RSM [25] Bacterial fibrinolytic enzymesespecially Bacillus sp can be effectively utilized to cure orprevent cardiovascular diseases [34] In the present studysequential optimization strategy was followed to enhancefibrinolytic enzyme production from Bacillus sp IND12 uti-lizing cow dung as the solid substrate

2 Materials and Methods

21 Screening

Step 1 (screening of protease-producing organisms) Soilfish and rice were used as the sample sources for the produc-tion of fibrinolytic enzyme Soil sample was collected from anagricultural field Rice was boiled for 45min and allowed foraerobic fermentation for a period of 48 h at room temperature(30 plusmn 2∘C) and used as the source of bacteria The marinefish Sardinella longiceps was collected from KanyakumariCoast Tamil Nadu India for the isolation ofmarine bacterialisolates One gram of sample was transferred to an Erlen-meyer flask (100mL) with 50mL of double-distilled water Itwas shaken for 10min and this was resuspended in distilledwater and aliquots were spread on skimmed milk agar plates(skimmed milk 10 beef extract 15 peptic digest of animaltissue 50 yeast extract 15 sodium chloride 50 agar 15bromocresol green 0015 [gL] pH 70) [35] For marineisolates 3 (vw) sodium chloridewas addedwith the culturemediumThe potent proteolytic enzyme-producing bacterialisolate was calculated on the basis of 119875119911 value as described byPrice et al [36] The bacterial isolate with low 119875119911 value wasconsidered for optimization of enzyme production119875119911 value was calculated by using the following equation

119875119911 =Colon diameter

Colon diameter + Zone of precipitation (1)

The isolated protease-producing bacteria were cultured indi-vidually in nutrient broth medium

This was performed by inoculating 1 (vv) preculturedprotease positive isolates and incubated at 37∘C for 48 h Thecell-free supernatant was used as the source of fibrinolyticenzyme

Step 2 (screening of fibrinolytic enzyme-producing bacterialisolates (secondary screening)) Fibrinolytic activity of thecrude enzyme was tested in a fibrin plate composed of 01Msodium phosphate buffer (pH 74) 1 (wv) agarose 12(vv) fibrinogen and thrombin (100 NIH unitsmL) Thefibrin plate was allowed to stand for 1 h at room temperatureto form a fibrin clot layer About 20120583L of crude enzymewas dropped into holes and incubated at 37∘C for 5 h Thefibrinolytic enzyme exhibited a clear zone of degradation offibrin around the well thus indicating its fibrinolytic activity[37] Ten organisms were screened and potent organism wasretained for this study The potent isolate was subjected tobiochemical characterization and 16S rDNA sequencing

Step 3 (fibrin zymography) Fibrin zymography was per-formed to determine fibrinolytic activity of protein having















119884 = 1205720 +sum120572119894119909119894119894

+sum120572119894119895119909119894119909119895119894119895

+sum120572119894119895119896119909119894119909119895119909119896119894119895119896

+sum120572119894119895119896119897119909119894119909119895119909119896119909119897119894119895119896119897

+sum120572119894119895119896119897119898119909119894119909119895119909119896119909119897119909119898119894119895119896119897119898

(2)




119884 = 1205730 +sum3

120573119894119883119894119894=1

+sum3

1205731198941198941198831198942

119894=1

+sum3

120573119894119895119883119894119895119894119895=1

(3)









11 IND12 13 14

15 16 17 18

19 20

(a)

11

C

IND12

1514

13

1920

18

17

16

(b)11

MM

14201

294366974

14201

294366974KDa

KDa15141312 19 20181716

(c)













0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Star

ch

Xylo

se

Sucr

ose

Con

trol

Mal

tose

Glu

cose

Treh

alos

e

Carbon sources ()

0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Beef

extr

act

Gel

atin

Con

trol

Yeas

t ext

ract

Case

in

Ure

a

Pept

one

Nitrogen sources ()

0100200300400500600700800900

1000

Enzy

me a

ctiv

ity (U

g)

MnC

l 2

Na 2

HPO

4

Na 2

HPO

4

Con

trol

Am

chl

orid

e

Ferr

ous s

ulfa

te

Sodi

um n

itrat

e

Calc

ium

chlo

ride







Run Moisture(A)

pH(B)

Sucrose(C)

Peptone(D)





Enzyme activity (119884) = +165488 + 180119860 + 8825119861

+ 21419119862 + 14131119863

+ 26588119864 + 21569119860119862

minus 9875119860119864 minus 24906119861119862

minus 16606119861119863 + 11525119861119864







+ 23425119862119863 + 14956119860119861119862

+ 11656119860119861119863 minus 1235119860119861119864

+ 7956119860119862119864 minus 10194119861119862119864

minus 41206119861119863119864 + 7937119862119863119864

minus 106119860119861119862119863 + 16444119860119861119862119864

minus 22813119861119862119863119864

minus 23362119860119861119862119863119864(4)




= +292144 + 34022119860 minus 14718119861 minus 54364119862




Sucrose(119861)






minus 588931198612 + 707191198622(5)




1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















119884 = 1205720 +sum120572119894119909119894119894

+sum120572119894119895119909119894119909119895119894119895

+sum120572119894119895119896119909119894119909119895119909119896119894119895119896

+sum120572119894119895119896119897119909119894119909119895119909119896119909119897119894119895119896119897

+sum120572119894119895119896119897119898119909119894119909119895119909119896119909119897119909119898119894119895119896119897119898

(2)




119884 = 1205730 +sum3

120573119894119883119894119894=1

+sum3

1205731198941198941198831198942

119894=1

+sum3

120573119894119895119883119894119895119894119895=1

(3)









11 IND12 13 14

15 16 17 18

19 20

(a)

11

C

IND12

1514

13

1920

18

17

16

(b)11

MM

14201

294366974

14201

294366974KDa

KDa15141312 19 20181716

(c)













0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Star

ch

Xylo

se

Sucr

ose

Con

trol

Mal

tose

Glu

cose

Treh

alos

e

Carbon sources ()

0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Beef

extr

act

Gel

atin

Con

trol

Yeas

t ext

ract

Case

in

Ure

a

Pept

one

Nitrogen sources ()

0100200300400500600700800900

1000

Enzy

me a

ctiv

ity (U

g)

MnC

l 2

Na 2

HPO

4

Na 2

HPO

4

Con

trol

Am

chl

orid

e

Ferr

ous s

ulfa

te

Sodi

um n

itrat

e

Calc

ium

chlo

ride







Run Moisture(A)

pH(B)

Sucrose(C)

Peptone(D)





Enzyme activity (119884) = +165488 + 180119860 + 8825119861

+ 21419119862 + 14131119863

+ 26588119864 + 21569119860119862

minus 9875119860119864 minus 24906119861119862

minus 16606119861119863 + 11525119861119864







+ 23425119862119863 + 14956119860119861119862

+ 11656119860119861119863 minus 1235119860119861119864

+ 7956119860119862119864 minus 10194119861119862119864

minus 41206119861119863119864 + 7937119862119863119864

minus 106119860119861119862119863 + 16444119860119861119862119864

minus 22813119861119862119863119864

minus 23362119860119861119862119863119864(4)




= +292144 + 34022119860 minus 14718119861 minus 54364119862




Sucrose(119861)






minus 588931198612 + 707191198622(5)




1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


11 IND12 13 14

15 16 17 18

19 20

(a)

11

C

IND12

1514

13

1920

18

17

16

(b)11

MM

14201

294366974

14201

294366974KDa

KDa15141312 19 20181716

(c)













0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Star

ch

Xylo

se

Sucr

ose

Con

trol

Mal

tose

Glu

cose

Treh

alos

e

Carbon sources ()

0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Beef

extr

act

Gel

atin

Con

trol

Yeas

t ext

ract

Case

in

Ure

a

Pept

one

Nitrogen sources ()

0100200300400500600700800900

1000

Enzy

me a

ctiv

ity (U

g)

MnC

l 2

Na 2

HPO

4

Na 2

HPO

4

Con

trol

Am

chl

orid

e

Ferr

ous s

ulfa

te

Sodi

um n

itrat

e

Calc

ium

chlo

ride







Run Moisture(A)

pH(B)

Sucrose(C)

Peptone(D)





Enzyme activity (119884) = +165488 + 180119860 + 8825119861

+ 21419119862 + 14131119863

+ 26588119864 + 21569119860119862

minus 9875119860119864 minus 24906119861119862

minus 16606119861119863 + 11525119861119864







+ 23425119862119863 + 14956119860119861119862

+ 11656119860119861119863 minus 1235119860119861119864

+ 7956119860119862119864 minus 10194119861119862119864

minus 41206119861119863119864 + 7937119862119863119864

minus 106119860119861119862119863 + 16444119860119861119862119864

minus 22813119861119862119863119864

minus 23362119860119861119862119863119864(4)




= +292144 + 34022119860 minus 14718119861 minus 54364119862




Sucrose(119861)






minus 588931198612 + 707191198622(5)




1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Star

ch

Xylo

se

Sucr

ose

Con

trol

Mal

tose

Glu

cose

Treh

alos

e

Carbon sources ()

0

200

400

600

800

1000

1200

Enzy

me a

ctiv

ity (U

g)

Beef

extr

act

Gel

atin

Con

trol

Yeas

t ext

ract

Case

in

Ure

a

Pept

one

Nitrogen sources ()

0100200300400500600700800900

1000

Enzy

me a

ctiv

ity (U

g)

MnC

l 2

Na 2

HPO

4

Na 2

HPO

4

Con

trol

Am

chl

orid

e

Ferr

ous s

ulfa

te

Sodi

um n

itrat

e

Calc

ium

chlo

ride







Run Moisture(A)

pH(B)

Sucrose(C)

Peptone(D)





Enzyme activity (119884) = +165488 + 180119860 + 8825119861

+ 21419119862 + 14131119863

+ 26588119864 + 21569119860119862

minus 9875119860119864 minus 24906119861119862

minus 16606119861119863 + 11525119861119864







+ 23425119862119863 + 14956119860119861119862

+ 11656119860119861119863 minus 1235119860119861119864

+ 7956119860119862119864 minus 10194119861119862119864

minus 41206119861119863119864 + 7937119862119863119864

minus 106119860119861119862119863 + 16444119860119861119862119864

minus 22813119861119862119863119864

minus 23362119860119861119862119863119864(4)




= +292144 + 34022119860 minus 14718119861 minus 54364119862




Sucrose(119861)






minus 588931198612 + 707191198622(5)




1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Run Moisture(A)

pH(B)

Sucrose(C)

Peptone(D)





Enzyme activity (119884) = +165488 + 180119860 + 8825119861

+ 21419119862 + 14131119863

+ 26588119864 + 21569119860119862

minus 9875119860119864 minus 24906119861119862

minus 16606119861119863 + 11525119861119864







+ 23425119862119863 + 14956119860119861119862

+ 11656119860119861119863 minus 1235119860119861119864

+ 7956119860119862119864 minus 10194119861119862119864

minus 41206119861119863119864 + 7937119862119863119864

minus 106119860119861119862119863 + 16444119860119861119862119864

minus 22813119861119862119863119864

minus 23362119860119861119862119863119864(4)




= +292144 + 34022119860 minus 14718119861 minus 54364119862




Sucrose(119861)






minus 588931198612 + 707191198622(5)




1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







+ 23425119862119863 + 14956119860119861119862

+ 11656119860119861119863 minus 1235119860119861119864

+ 7956119860119862119864 minus 10194119861119862119864

minus 41206119861119863119864 + 7937119862119863119864

minus 106119860119861119862119863 + 16444119860119861119862119864

minus 22813119861119862119863119864

minus 23362119860119861119862119863119864(4)




= +292144 + 34022119860 minus 14718119861 minus 54364119862




Sucrose(119861)






minus 588931198612 + 707191198622(5)




1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Sucrose(119861)






minus 588931198612 + 707191198622(5)




1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1000

100090

080070

060050 9000

950010000

11000


10500

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

(a)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010009 008 007

009 008 007006006 005 9000

950010000

11000

A m

oisture

()10500

C MgSO4 ()

(b)

1000

2000

3000

4000

5000

6000

Enzy

me a

ctiv

ity (U

g)

010010 009 008 007009 008 007 006006 005C MgSO4 ()

B s

ucro

se (

)

100090

080070

060050

(c)









4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4 Conclusion


Competing Interests




Acknowledgments


References











































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

novel sequential screening and enhanced...

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