© 1978 HASSAN DAL I LOTTOJ ARI ALL RIGHTS RESERVED AN EVALUATION OF SELECTED TECHNIQUES FOR REDUCTION OF NUCLEIC ACIDS IN PROTEINS OBTAINED FROM SACCHAROMYCES CEREVISIAE BY Hassan Dalilottojari A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1978 (Ema-8m ABSTRACT AN EVALUATION OF SELECTED TECHNIQUES FOR REDUCTION OF NUCLEIC ACIDS IN PROTEINS OBTAINED FROM SACCHAROMYCES CEREVISIAE BY Hassan Dalilottojari Methods for decreasing the nucleic acids (NA) of disintegrated yeast cells and their effect on the isolated proteins were investigated. The methods used consisted of: l. Precipitation of the NA with protamine sulfate, streptomycin, manganous chloride and/or phase— separation. The optimum concentration of protamine sul- fate for maximum reduction of NA and minimum loss of protein was determined in which nucleic acids were reduced up to 64 percent with protein loss of only 50 percent. Samples treated with protamine sulfate showed more protein zones on different electrOphoretic techni- ques than any other treatment. Reduction of NA by pro- tamine sulfate caused an increase in total sulfhydryl and available lysine in the proteins. Streptomycin reduced the NA up to 57 percent while protein recovery was 76 percent. MnC12 caused precipitation of NA up to Hassan Dalilottojari 55 percent. Protein loss was only 30 percent. Storage of the treated samples at 0°C resulted in additional precipi- tation of the NA. With polyethylene glycol and dextran, up to 66 percent of the total NA could be separated. The molecular weights of the proteins after removal of NA were measured on gradient SDS-polyacrylamide gel electrOphoresis (PAGE). Isoelectric points of most of the proteins were in the range of 4.5 to 7.5. Proteins remaining after these treatments were generally unde- natured, since the activities of lactic dehydrogenase and esterase were retained. 2. Separation of proteins from NA by precipita- tion. This was accomplished by hot sodium chloride or pH adjustment. The protein loss was very significant, but the ratio of proteins to NA was higher than with other methods of separation. Most of the proteins remaining were denatured. Electr0phorograms of the proteins exhibited very few zones with different mobility from control. 3. Hydrolysis of NA. Activation of endogenous ribonuclease (RNase) by a heat-shock followed by incuba- tion caused reduction of NA up to 56 percent. Treating yeast cells with bovine RNase lowered the NA to 39 per- cent. Protein loss was significant in both methods. Heat treatment apparently caused denaturation of the pro- teins. Proteins were affected more by heat-shock than Hassan Dalilottojari incubation. 0n electrophoresis, the exogenous RNase- treated sample showed fewer zones than the proteins obtained by endogenous RNase. Molecular weight of the proteins in exogenous RNase method were in the range of 14,300 to 318,000 while in endogenous RNase were in the range of 13,000 to 212,000. Proteins remained after the removal of NA had generally higher levels of essential amino acids than the untreated sample. 4. Removal of NA by affinity chromatography. On a DEAR-cellulose column, most of the proteins were sepa- rated from NA. Part of the proteins were eluted along with NA which could be detected on PAGE of the fractions. Protamine was immobilized on Sepharose and used for separation of NA. Addition of 20 percent dioxane to the elution buffer improved the separation. Removal of NA from a freeze-dried yeast homogenate was more diffi- cult. By a column of immobilized RNA on Sepharose, part of the NA (less than 10 percent) was separated. Yeast proteinases were investigated in another study. They behave like both chymotrypsin and trypsin. Phenylmethyl sulfonyl fluoride inhibits the activity of these proteinases to a large extent. Soybean trypsin inhibitor at concentrations of about 1001K! per mg yeast protein has strong inhibitory effect on yeast proteinases Hassan Dalilottojari while navy bean trypsin inhibitor even at higher concen- trations did not have any significant effect. Soybean trypsin inhibitor immobilized on Sepharose removed a significant quantity of the proteinases. ACKNOWLE DGMENTS The author wishes to express his sincere appre— ciation to his major professor, Dr. C. M. Stine, for his assistance throughout this study. Acknowledgment is extended to the other members of the guidance committee, Drs. P. Markakis, Professor of Food Science; H. A. Lillevik and L. L. Bieber, Pro- fessors of Biochemistry, for reading this manuscript. Thanks to Dr. Wood and Ms. D. H. Bauer (Bio- chemistry Department) for help and performing amino acid analysis for this study. Special thanks are due Dr. J. R. Brunner and to the author's fellow graduate students for their many contributions. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . The Potential Utilization of Single Cell Protein . . . . . . . . . . . Other Uses of Yeast . . . . . . . . The Yeast Cell Wall . . . . . . . . Techniques for Breaking the Cell Wall of Yeasts . . . . . . . . . Enzymatic Hydrolysis . . . . . . . High Pressure Homogenization . . . . Ultrasonic Vibration . . . . . . . Agitator Mill . . . . . . . . . Ball-Mill Disintegrator . . . . . . Nucleic Acids in Yeasts . . . . . . Techniques to Lower the Nucleic Acid Content . . . . Removal of Nucleic Acids by MnC12 . . Removal of Nucleic Acids by Phase Separation . . . . . . . Removal of Nucleic Acids with Protamine Sulfate . . . . . . . . Removal of Nucleic Acids by Streptomycin Sulfate . . . . . . . . . Lowering the Nucleic Acid Content by Protein Precipitation . . . . Removal of Nucleic Acids by Ribonu- cleases . . . . . . . . Removal of Nucleic Acids by Base Hydrolysis . . . . . . . . . Affinity Techniques in Removing Nucleic Acids . . . . . . . . . . . . Proteinases . . . . . . . . . . iii Page vi viii UlU'IUJ Hru oc: flu9mmfiflEOunTcwmocm>UIl.oa musmflm 48 manomo 02.24] wkdzommdoooi. IOL ZMUIOJ xuodzm mm wmomdxaum mmomded meJDJJNU {A 1 .3 £20 to w 49 2.6 x 40 cm column. Before use, the Sephacryl was equilibrated with the desired buffer. RESULTS AND DISCUSSION I. Effect of Removing Nucleic Acids on Proteins Breaking the resistant wall of yeasts increases the amount of protein that can be extracted from the cell. Mechanical disintegrators have an advantage over other procedures in that they result in less protein denatura- tion. Therefore, in the course of this study a mechanical homogenizer was used to disrupt the cell. Several different methods were employed and eval- uated to obtain yeast protein low in nucleic acids (NA). Since ribonucleic acids (RNA) are the dominant nucleic acid, adequate reduction Of RNA content is sufficient for protein to be used for food purposes. Effect of the techniques of removal of NA on the remaining proteins will be evaluated, i.e., protein recovery, extent of denatura- tion of proteins and changes in amino acid composition of the whole protein fraction. Finally, by using dif- ferent electrophoretic techniques, the effect of NA removal on the proteins will be evaluated in more detail. 50 51 MnClz Precipitation of Nucleic Acids The addition of 0.1 M MnCl2 (5 percent v/v) to the cell homogenate caused precipitation of NA which were readily separated from the protein fraction by centri- fugation. A portion of the proteins was also removed. In the experiment done, NA content was lowered from 0.26 percent to 0.12 percent (55 percent removal) and protein was lowered from 2.40 percent to 1.66 percent (30 percent loss, Table 1). Using MnCl2 for the separation of pyruvate enzymes, Korkes et al., (1951) reported a large loss of the enzymes. Mn2+ is one of the cations which is able to alter the solubility of NA and results in their precipitation. It is relatively cheap and does not interact with all of the proteinaceous components, i.e., in polyacrylamide gel (Figure 11, 2) esterase were detected while lactic dehydrogenase was not. If the resultant protein is intended for use as food, the toxic manganese must be removed by dialysis or filtration. Storing the treated sample on ice resulted in still more precipitation of the NA. After five days when the precipitate was removed by centrifugation (17000 x g for 20 minutes), the NA content was reduced to as low as 0.03 percent (from 0.12 percent or 11 percent of the original). However, the protein content was 52 TABLE l.-~Effect of different treatment on removing nucleic acids and protein recovery % Protein % Nucle1c Protein, Treatment Recovery 22mgved Egglgic Acid Control 100 0.0 9.0 2/10 v/v of 2% protamine sulfate 72 42 12.4 Phase separation 50 66 11.1 MnCl2 70 55 13.9 Streptomycin 76 57 11.3 pH adjustment 19 79 14.5 NaCl precipitation 40 63 22.8 Exogenous RNase 61 63 15.3 Heat shock 75 56 13.0 53 lowered to 0.42 percent (from 1.66 percent or 17.3 percent of the original). The polyacrylamide gel electrophoresis (PAGE) pattern of MnCl2 treated yeast protein was very similar to the untreated sample except the protein zones at the lower level of the gel (Figure 12, 5) were not easily detected. The reason could be that the more acidic pro- teins are more susceptible to precipitation. In SDS (sodium dodecyl sulfate)-PAGE, in which the proteins are broken to their constituent subunits and they separate according to their molecular weight, the proteins of the MnCl2 treated sample had molecular weights in the range of 14,000 to 200,000 (Figure 13, 6). In isoelectric focusing (Figure 14, F), the iso- electric point of the proteins can be compared with that of the control (in this experiment, ampholyte pH range 5-8 was used). Most of the proteins had pI in the range of 4.5-7.5. There could be more proteins detected in the treated sample than in the control, but results were com- parable. Phase Separation of Nucleic AOids Dextran and polyethylene glycol, when used in apprOpriate concentrations, will cause precipitation of NA. In this experiment the nucleic acid content in the 54 Figure ll.--Polyacrylamide gel electrophorogram of yeast proteins stained for lactic dehydrogenase and esterase l-untreated sample, 2—MnC12 3-protamine sulfate + proteinase inhibitor (PMSF) 4-protamine sulfate no proteinase inhibitor, 5-phase separation, 6-streptomycin, 7-protamine sulfate + proteinase inhibitor stained with Coomassie Brilliant Blue G-250. Arrows indicate bands which display either lactic dehydrogenase or esterase enzymatic activity. O 0 O O r» , Intr- I 2 3 55 56 Figure 12.--Polyacry1amide gel electrOphorogram of yeast proteins in 7.5-17.5 percent w/v gels after different treatment for reduction of nucleic acids: l-control, 2—exogeneous RNase, 3-heat- shock, 4-pH adjustment, 5-MnC12, 6-protamine sulfate + proteinase inhibitor (PMSF), 7-protamine sulfate no proteinase inhibitor, 8-NaCl, 9-phase separation, lO-streptomycin, ll-commercial spray-dried yeast. 57 . .3 II... 9 IO 8 7 58 .cflo>aoumwuumlm .cofiumummom mmmnmlm .mumuanm wcflEmuoumlh .maoczum .ucwfiumsmpm mmum .HOMZIe .xoonmIDMOSIm .mmmzm msocmmoxmlm .HOHDGOOIH .uwma may :H Ozonm mum mumxumfi unmflmz HMHOOOHOE mom an OmsHEHmumO mm mcflwuoum mnu mo unmflm3 unasomaoz .mawm ucmflpmum A>\3v usmonmm m.halm.b Ga mcflwuoum ammo» mo mcnmuumm Oflumuosmouuomam mom on» no Emnmmw0|l.ma musmwm IIIIIIBDIIEIII II '1! I 9. 9. 59 I + A a O a! C fi I l 903. "103310" x0. to 60 .ume can OH csocm ma pwEHOm ucmflpmum mm .mpmmasm OCHEmuoudlm .coflumnmmwm mmmnmlo .NHUQEIm .ucmEumsmOm mmlm .cflo>EOummHumlo .xoonmuumwnlu .wwmzm msocmmoxmlm .Houucoon< Amlm may msamsOOM OHHuomeomH :H cflwuoud ammo» mo mcumuumm owumnonmouuowam may mo Emummwall.va musmflm rj IlIi‘ i I I 61 IIIIEII “III 2 4 I . EN O.m On . v6 . 0.? . _.m . m6 m6 . Em . Qm 1 m6 . Wm . 06 . 0.x. . m4: 62 homogenized yeast cell was lowered from 0.26 percent to 0.09 percent (66 percent reduction) and protein loss was from 2.4 percent to 1.2 percent (50 percent) (Table 1). The sodium chloride added during this technique is some- what unattractive in that its subsequent removal would entail additional expense in a commercial process. The cost of polyethylene glycol and dextran is another econ- omic limitation to the method unless they can be recovered. The loss of protein can be attributed to NaCl and complexes formed between proteins and polyethylene glycol. Advantages of this technique are that it does not affect Uuaproteins and thus can be used for isolation of enzymes (Garret et al., 1969). This was not proved when gels were stained for lactic dehydrogenase and esterase (Figure 11, 5). In PAGE, the number of protein zones detected were comparable with that of the control except for a few at the lower level of the gels (Figure 2, 9). In SDS-PAGE (Figure 13, 8), most of the proteins showed different mobility from that Of control. This might be from retained traces of polymer attached to the proteins. Proteins which could not get into the gel and remained on the top supports this possibility. In iso- electric focusing, there was a poor resolution (Figure 14, G). Most of the proteins did not move below pI range of 6.9, and this is another evidence for the 63 polymer-protein complex left into the solution mentioned before. Protamine Sulfate as Nucleic Acid Precipitating Agent Protamines which are basic proteins (high in arginine) are capable of binding to NA and promote their precipitations. Highly acidic proteins will be removed along with precipitation of NA, and this accounts for protein loss. The more protamine sulfate added to the cell homogenate, the more NA will be removed but with consequent losses in protein, as can be seen in the data presented in Table 2. The optimum concentration of protamine sulfate was obtained when four volumes of a 2 percent w/v protamine sulfate solution (80 mg protamine sulfate) was added to ten volumes of cell homogenate (1.02 g yeast protein). This resulted in a 64 percent reduction of NA and only a 50 percent loss of protein. Higher concentrations of protamine sulfate such 160 mg per 1.02 g yeast protein (8/10 v/v) only reduced the NA 77 percent. In other words, by doubling the pro- tamine sulfate concentration, the NA reduction increased only 13 percent. Protamines have no adverse effect on the yeast proteins which makes them excellent for purification of enzymes. Lactic dehydrogenase and esterase could be readily detected in gels (Figure 11, 3 and 4). 64 A>\3 unmoumm NV m.a~ he be we mummasm mcwemuoum mo >\> oa\m A>\3 unmoumm NV «.ma be we be mummasm mcflsmuoum mo A>\>V oa\m A>\3 usmoumm NV N.OH «O we om mummasm OGHEmuOHm no x>\>c oa\e A>\3 unmoumm NV >.oa ma mm mm mummasm OCHEmuonm mo 1>\>O oa\~ A>\3 ucwuumm NV m.oa m mm hm mummasm msflfimuoum mo A>\>v oa\a m.m o.o OOH ooa ucmfiummuu oz Oflumm Hm>OEOm >Hm>oomm >Hm>wowm pflom OAOHODZ UHO< OAOHODZ :wmuoum Umaom :Hmuoum ucmoumm unwoumm H p B . unmouwm pfiom Owoaosc unwoumm OH.H tam cflmuoum unmoumm N.OH .OHHOm Hmuou uamoumm vm.ma Umcflmucoo mumcmmOEon HHOO mcwuumum .>HO>OOOH CHODOHQ tam OHHOm Hmuou 0cm mpflom camaoss mo Hm>OEOH so mumMHSm OCHEODOHQ mo mcoflumuucwocoo pcOAOMMHO mo HOOMMMII.N mqm<9 65 Samples treated with protamine sulfate showed more protein zones (by electrophoresis) than any other treatment, even the control sample exhibited fewer com- ponents (Figure 12, 3 and 4) which may be due to the removal of NA which otherwise associate with proteins. In SDS-PAGE, proteins showed molecular weights in the range of less than 10,000 up to 200,000 or more (Figure 13, 7). In isoelectric focusing (Figure 14, H), pro- teins had isoelectric points ranging from 3.2 to 7.5. Reduction of NA by protamine sulfate increased total protein sulfhydryl content as shown in Table 3. Available sulfhydryl group has a slight decrease in reduced NA sample. Addition of proteinase inhibitor increases both available and total sulfhydryl group. A similar increase was noted for available lysine (Table 4). Overall nucleic acid removal with protamine sul- fate was quite satisfactory. As protamine sulfate is relatively expensive ($45/lOOg), recovery and reuse would be important. One way to accomplish this is by immobilization, which will be subsequently discussed. Streptomycin as a Nucleic Acid Precipitating Agent Streptomycin, which is a positively charged NH moleculelbecause of guanidino radical (-—NH——C——NHH 66 TABLE 3.--Effect of removing NA by 2/10 v/v of a prota- mine solution (2 percent w/v) and proteinase inhibitor (PMSF) on sulfhydryl and disulfide group of yeast proteins Treatment SH(uM/g Protein) SS(uM/g Protein) Cell homogenate 28.2 39.0 Protamine sulfate treated, no inhibitor 23.0 42.3 Protamine sulfate treated, + inhibitor 33.1 49.7 TABLE 4.--Effect of removing NA by 2/10 v/v of a prota— mine sulfate solution (2 percent w/v) and proteinase inhibitor (PMSF) on available lysine in yeast proteins Treatment g lysine/100 g protein Cell homogenate 3.3 Protamine sulfate 3 8 treated, no inhibitor ' Protamine sulfate 4 1 treated, + inhibitor 67 can bind to NA and lower their solubility. In the experi- ment reported here, 0.1 M streptomycin at ratio of 1:16 to protein, lowered the NA content up to 57 percent while protein recovery was 76 percent and the ratio of protein to NA was raised to about 11.5 (from 9.1). In PAG, the streptomycin treated sample showed fewer zones at the lower part of the gel (Figure 12, 10). When the gels were stained for lactic dehydrogenase and esterase, the activity of the enzyme could be detected (Figure 11, 6). Apparently, this method of lowering NA does not denature the proteins so that it can be used for isolation of enzymes or where undenatured protein is needed for some functional use. SDS-PAG pattern of strep- tomycin treated samples (Figure 13, 9) looked much the same as the control except for a few zones which had disappeared at the bottom of the gel. This could be due to reaction of more acidic proteins with streptomycin which is a positively charged compound and as a result precipiation of these proteins along with NA. In isoelectric focusing (Figure 14, D), the pro- tein zones were quite close and similar to untreated sample. Most of the proteins had isoelectric points in the range of 3.7 to 7.5. Streptomycin is relatively inexpensive but recovery would again be important if it is going to be used in large scale. Streptomycin is not 68 permitted in foods under present regulations, and this also would necessitate quantitative removal and recovery. pH Adjustment Jayaraman (1973) suggested that at pH 3.9 most proteins could be separated from nucleic acids. In this research, cells were homogenized in 0.1 M phosphate- citrate-borate buffer pH 7. The proteins were then pre- cipitated by lowering pH of the buffer to 3.9 and removed. Recovery of the proteins in this method was very low (only 19 percent), but 79 percent of the NA were removed (Table 1). In PAGE, fewer protein zones could be detected and most of the proteins showed had different mobilities (Figure 12, 4). Lactic dehydrogenase and estrase could not be detected in the gels which may be from denatura- tion or more likely to the poor recovery of protein. SDS-PAG (Figure 13, 5) showed very few protein zones and these were in the molecular weight range of 20,000 to 150,000. Isoelectric focusing (Figure 14, E) showed the proteins to have pI in the range of 4.5 to 6.5. This method as evaluated indicated very poor pro- tein recovery because of incomplete precipitation at pH 3.9. It has the advantages of being simple, inexpensive and rapid. 69 Separation of Yeast Proteins with Sodium ChIOride The proteins were precipitated in the presence of 10 percent (w/v) NaCl and the NA remaining in the solu- tion were separated by centrifugation. The heat employed and the salt are strong enough to cause extensive alteration to the proteins as indi- cated by loss of enzymatic activity when protein zones separated by electrophoresis were checked for activity. Protein recovery (40 percent) was not satisfactory under the conditions employed, but 63 percent of the NA were removed (Table l), yielding the highest protein/NA ratio of the methods used. In PAG (Figure 12, 8), very dew faint zones could be seen. Aggregated proteins which could not get into the gels remained on the top. In SDS-PAG (Figure 13, 4), SDS and mercaptoethanol disaggregates these proteins so more zones were Observed. The proteins had molecular weights in the range of 16,000 to about 210,000. In isoelectric focusing, because of aggregation of the pro- teins, not any zone could be detected while top of the gel was heavily stained. The problems associated with this technique were poor protein recovery, denaturation of proteins and salt removal. Advantages of the method are speed, low cost and the high protein NA ratio. 70 Exogenous Ribonuclease for Reduction of Nucleic Acids Bovine pancreatic ribonuclease A (RNase A), which is commercially available, can be used to hydrolyse ribo- nucleic acids (RNA). RNase A is an endoribonuclease,iue., hydrolysis of phosphodiester groups can occur anywhere in the chain. It is specific for single stranded RNA and has absolute specificity for pyrimidine nucleotides. To the heat-shocked yeast cells, RNase A was added, the mixture incubated, then centrifuged. The cells depleted of NA were then homogenized. The data in Table 5 show the effect and importance of each step in removing NA. Incubation (at 55°C for 60 minutes) helped depleting the cells of NA (three times more NA was leaked out of the cell compared to not incubated sample). Heat- shock is very effective in facilitating penetration of RNase into the cell. It also activates the RNase (Castro et al., 1971). Heat-shock increased reduction of NA five-fold more than not heat-shocked sample as can be seen in Table 5. Addition Of RNase helped lower NA, but when compared with heat-shocked and incubated sample (NA breakdown is from endogenous RNase), the difference was not significant. Protein recovery by this method was 61 percent and NA removal 63 percent and protein NA ratio of 15.3 (Table 1). The main reason for protein loss may well 71 TABLE 5.--Effect of different treatments withoo uoooouo coon .nooH ..o .pmppm uouwnqccfi OmwcwouOHQ o: pwummuu .pmusmmos uoc mm: m wumuaam onwamuoum N munuasm wcaswuoum H .m .owsuoooz scum. 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S U .4 p. 3. 3 O u A u. 0 m o 1 a T. a . 1. two: ..a o n m 1. .cu .lu e o s u o a a ocafid S X. 3 D U . .4 m... .cfimuoum pouoouuoo O OOH\pwoc ocwfim my mucmfiummuu ucmuomuac zn mpwom Owwaosc ca pmuosoa mchuoaa umww> mo ceauwwomeoo vwom OswE How memzm sump nouns m CH Oohm um Omumnsocfl mumcwmoeo: HHOO ummwm mo cumuumm Owumuonmouuowam mo EmHOMHQII.mH musmflm 97 I min 60 III II I; )9, II I I :7! I; 2 I II I I g; I I” IIII IIII'IIII II U: T ’I I I 98 .mmEHu maoflum> How memcw came Hmum3 m OH Uohm um pmumnsosfi wumswmoaon HHOO ummm> mo cumvumm oeumuonmouuomam mo Emummaouu.om musmflm 99 I.) i +3 IILIII I T 12 I III I I J: IIILLI I I2 1 1le I I I H. L" I’FJTII I. I 1 III I. II» L’IIILL III I I . - I[IILLL l I .. L' ff JLL‘: I II; ’IIEIIL II I II» I E‘ITII I I3 ’ -- II IIIIII n- WIIIII I III II II I 7. 100 .cofludaom xooum mm pom: mm: cflmuoum OE hm nae3 Amabmpcmfiflpmmcocv mumcmmOEon HHOO ammo» mo HE mco .AOBmHV xooaflz can Swan: 0» mCHOHOoom .>Hm>fluoommmu .8: mmm paw new um mocmnuownm :fl wmcmco mm pmusmmmE AmmE>Ncm aflmmhuu08>so paw cflmmhuu How Demaommm mmumuum neon ouflwcucmmv mmam 0cm m2¢9 co mommcflmuoum ammo» mo cOwuo¢II.Hm musmflm 101 amuuz>Nzu mg... z_mm>¢h ....... o ......... AmvuZ>Nzw ux... 25153.0 1»... oIIIoIII 0.x. ON 20:31.5 0? OIX (NIW/aonvaaosav v) 3" S: N N cu 102 Figure 22.—-Densitometric tracing of yeast proteins on gradient SDS-PAG (7.5-17.5 percent w/v). A is homobenated yeast cell B is yeast protein low in nucleic acid (treated with 2/10 v/v of a 2 percent protamine sulfate) + proteinase inhibitor (PMSF) C is same as B but no inhibitor 104 Figure 23.—-Densitometric tracing of yeast proteins on 7.5 percent w/v PAG. A is homogenated yeast cell + proteinase inhibitor (PMSF) B is yeast protein low in nucleic acid (treated with 2/10 of a 2 percent protamine sulfate) + PMSF C is same as B but no inhibitor 106 .mfimxaowuoum on map commas mmcoN samboum "muoz .ucmaumwuu ou0mwb poppm mm3 mmzm mo Aaocmnumv HE\OE m o no >\> om\HIm nonfibflncfl mmmcflmpoud mam m>mn Doc mmopld (.mpflum OHmHosc on» manuamflomum Ou HOOHO Ga ovumasm mcflfimuoum unwound m o no >\> oaxvuo .>\> oa\~-m .>\> oH\H suflz omuoonuIN .Omummuucnla .pmma on» CH CBOcm ma unmflmz umHSOOHOE paw mufiHAQOE m>flumamu cmmzumn mflnmcoflumHmu one .o\3v usmonmm m.>H|m.h cfl Apmummuu mummasm msflsmuoumv Uflom Demand: :H 30H mcflmuoum ammo» mo mcumuumd oflumuonmonuomam may mo Emummflaln.¢m musmflm 107 l . l [:1 _I I IIIIII: I I I III . :ITI‘H MT 0 .I .1?” 1° lelIIIIIIIIIImII III 1m do? II I [III I * 2.0IX lHBIBM UV‘II'IOB‘IOW O 000 g; cove r~ ES 3 a w '6 o I: . " O G ° 3 Q <5 o 8 2 a AIIWIBON 3M1V13 108 in ethanol), exhibited more zones than the sample without gut PMSF. The data in Tables 3 and 4 show total disulfide, available sulfhydryl and available lysine were more preva- lent in yeast proteins contained PMSF as proteinase inhibitor than in the controls. Sulfonyl halides such as PMSF react with many serine proteinases to sulfonylate the hydroxyl group of a specific serine residue in the active site and thereby inhibit enzymatic activity (Gold, 1967). Yeasts contain a variety of proteolytic enzymes. Hence, it is quite difficult to eliminate all the proteoly- tic artifacts by using one inhibitor. Another problem in using the inhibitors is the extreme toxicity of most of these compounds. Therefore, in anther study, effect of some of the naturally occurring antitryptic compounds on these proteinases was evaluated. Navy bean trypsin inhibitor isolated from Phaseolus vulgaris L., which is an albumin, even at concentrations of 1:3 to total yeast proteins had no or very small inhibitory effect on yeast proteinases (Table 8). Navy bean trypsin inhibitor interacts strongly with trypsin as well as chymotrypsin (Gomes, 1978). Soybean trypsin inhibitor (Kunitz) showed strong inhibitory effect on yeast proteinases as can be seen from 109 TABLE 8.-—Effect of navy bean trypsin inhibitor (N.B.T.I.) on yeast proteinases measured by method of Kakade et al., (1969) Concentration pg N.B.T.I./3.3 mg Protein Absorbance at 280 nm 0-0 0.380 100 0.330 300 0.360 500 0.330 800 0.375 1100 0.370 110 the data in Table 9. Percent inhibition was maximum at 300 ug soybean trypsin inhibitor. At higher concentra- tions Of the inhibitor, the inhibition was reduced. Soybean trypsin inhibitor, which is a globulin, has molecular weight of 21,500. It consists of a single polypeptide chain. At neutral pH, it forms a one-to-one stoichiometric complex with trypsin while appreciable interaction also occurs with chymotrypsin (Steiner and Frattali, 1969). Because of strong inhibitory effect of soybean trypsin inhibitor on yeast proteinases, immobilized soy- bean trypsin inhibitor on Sepharose 4B was used to remove the proteases from yeast proteins. When 30 mg of yeast protein was applied on a column (0.5 x 12 cm) packed with soybean trypsin inhibitor-//—Sepharose 4B (Figure 25), more than 50 percent of proteasess were absorbed by the column which were later eluted by 0.1 M acetate buffer (pH 3.0) under the second peak. By this method, it is not only possible to mini- mize the problem of proteolysis, but also prepare puri- fied proteases for other purposes. There are several other compounds which have affinity toward the proteinases and might be used in immobilized form to remove the reamining proteinases. Aminocaproyl-p-amino-benzamidine which binds to several 111 TABLE 9.--Effect of soybean trypsin inhibitor (S.B.T.I.) on yeast proteinases measured by method of Kakade et al., 1969 u g S.B.T.I./3.3 mg Absorbance/ Percent Inhibition Protein 280 nm 0.0 0.491 0.0 100 0.115 76 200 0.110 78 300 0.075 85 400 0.073 85 500 0.070 85 800 0.107 78 1000 0.195 60 1500 0.320 35 112 .xmmm paoomm may Hmpa: pmysam tam aEsHOO may Oy pmaHOmam mymz mmmmaflmyoym mo yamoymm om amay myoz .NHOmO yam om pmaflmyaoo aowa3 o.m am Hmmmsa ways 2 H.o ma GEDHOO Eouw pmysam AmyMMHSm mcHEmyoym yamoymm m m mo >\> OH\¢ ayys pmymmyyv OHom Oflmaosa :H 30H aymyoym ymmmh mE om .AEO NH x m.ov afisaoo mv mmoymammml\\syoyflaflaafl sflmmhyy cmmamOm m so mmmmcfimyoym ymmm> mo mammymoymfioyao Myycwmmanl.mm mysmflm 113 mmmZDz 20:. 04mm ON 0. O. o |II r H 1/ 0.0. u IdI /O / LAV/o '0. O 1 °. 1 '8. u“’08? BONVBUOSBV 6? N 114 different type of proteinases (Jany et al., 1976) and glycyl-L-tyrosine-azo-benzyl-succinic acid which is specific for carboxypeptidase Y and A (Johansen et al., 1976) are examples of these compounds which can be used. SUMMARY Commercial press cake of baker's yeast was washed and disintegrated in a cell homogenizer. A detailed study was made of different techniques to lower the nucleic acid content of the isolated yeast proteins. These tech— niques can be categorized into three major groups: 1. Precipitation of either proteins or nucleic acids 2. Enzymatic hydrolysis of nucleic acids, and 3. Affinity chromotography The effect of each process on the separated pro- teins was studied. Protein yield, amino acid composition, extent of denaturation, electrophoretic characterization and effectiveness of each method in nucleic acid removal were of major interest. Electr0phoretic characteriza- tion included isoelectric focusing, disc gel electro- phoresis and SDS polyacrylamide gel electrOphoresis. Each method for removing nucleic acids has some advantages and some limitations. Protamine sulfate pre- cipitation was found to have no obvious effect on the proteins. Hot NaCl precipitation denatured the majority of the proteins. Enzymatic methods resulted in proteoly- sis and protein denaturation. These techniques are 115 116 comparable as regards to nucleic acid removal. Selection of the method to be used will be determined by economical feasibility, use and fate of the proteins, i.e., if undenatured protein is-needed for texturized protein products or commercial enzyme production. The native proteinases present in yeasts are a serious problem because they result in loss of protein and change in protein characteristics with time. There- fore, in another study the effect, control and removal of these proteinases was examined. Yeast proteinases behave more like chymotrypsin rather than trypsin. Phenylmethyl sulfonyl fluoride can be used to inhibit proteinases in routine laboratory work. Soybean trypsin inhibitor at concentrations of about 100 pg per mg yeast protein has strong inhibitory effect on yeast protein- ases while navy bean trypsin inhibitor does not have any significant effect. Soybean trypsin inhibitor was immo- bilized on Sepharose 4B and was used to remove a signifi— cant quantity of the proteinases. Proteinases once separated can be recovered and serve as a source of proteolytic enzymes for appropriate uses in the food industry. APPENDIX 117 APPENDIX TABLE A.1.--Some important chemicals used in this study and their sources Chemicals Sources Acrylamide Alcohol dehydrogenase Alkaline phosphatase N-benzoyl-L—tryrosine ethyl ester Glucose oxidase Insulin Invertase B—Lactoglobulin N-N'-methylene-bis- acrylamide Navy bean trypsin inhibitor Phenyl methyl sulfonyl fluoride 6-Phosphogluconic dehydrogenase 118 Bio-Rad Laboratories Richmond, CA Sigma Chemical Co. St. Louis, MO Sigma Chemical CO. St. Louis, MO Sigma Chemical CO. St. Louis, MO Worthington Freehold, NJ Sigma Chemical Co. St. Louis, MO Sigma Chemcial Co. St. Louis, MO Nutritional Bio Chemi- cal Corporation Cleveland, OH Bio-Rad Laboratories Richmond, CA Carlos Gomes Dept. of Food Science M.S.U. E. Lansing, MI Sigma Chemical Co. St. Louis, MO Sigma Chemical CO. St. Luois, MO 119 TABLE A.1.--Continued Chemicals Sources Phosphoglucose isomerase Protamine sulfate RNA SDS Sepharose Soybean trypsin inhibitor-//-Sepharose Streptomycin sulfate TEMED p-toluenesulfonyl-L-arginine methyl ester Sigma Chemical Co. St. Louis, MO Sigma Chemical Co. St. Louis, MO Nutritional Biochemi- cal Corporation Cleveland, OH Pierce Rockford, IL Pharmacia Fine Chemi- cals Uppsala, Sweden Sigma Chemical Co. St. Louis, MO Sigma Chemical Co. St. Louis, MO Bio-Rad Laboratories Richmond, CA Sigma Chemical Co. St. Louis, MO 120 TABLE A.2.--Formulation of 7.5 percent Polyacrylamide Gel Electrophoresis System Separating Gel (7.5% Acrylamide, 0.18% BIS, pH 8.9) Stock solutions amount /100 ml a) Tris 36.3 g N,N,N',N'-Tetramethylethylene- diamine (TEMED) 0.23 ml b) Acrylamide 30 g N,N'-Methylenebisacrylamide (BIS) 734 mg c) Ammonium persulfate 140 mg Working solution - 1 part (a):l part (b):2 parts (c) Stacking Gel (2.5% Acrylamide, 0.625% BIS) Stock solutions amount/100 m1 a) Tris 5.98 g TEMED 0.46 m1 1 N HCl to yield pH 6.7 b) Acrylamide 10 g BIS 2.5 g c) Riboflavin 4.0 mg d) Sucrose 40 9 Working solution - 1 part (a):2 parts (b):l part (c): 4 parts (d) Buffer Solution (0.04 M Tris, 0.2 M Glycine, pH 8.3) 23./.1 Tris 3.0 Glycine 14.4 REFERENCES 121 REFERENCES Afting, E. G., Lynen, A., Hinze H. and Holzer, H. 1976. Effect of yeast proteinase A, proteinase B and carboxypeptidase ycmuyeast phosphofructokinase. Hoppe-Seyler's Z. Physiol. Chem. 351:1771. Aibara, S., Hayashi R. and Hata, T. 1971. 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