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Protein in Lemna minor L.
Biochem. Physiol. Pflanzen (BPP), Bd. 167, S. 105-108 (1975)
Short Communication
Protein in Lemna minor L. W.
MACIEJEWSKA-POTAPCZYK,
L.
KONOPSKA
and
K. OLECHNOWICZ
Department of Plant Physiology, Institute of Physiology and Cytology, University of Lodz, Lodz, Poland Key Term Index: bulk protein, amino acid composition, nutritional value; Lemna minor.
Summary By control of growth conditions a continuous culture of Lemna minor was obtained. Total protein in Lemna minor corresponds to 16 % of dry matter. The fraction elutable with 0.015 M orthophosphate buffer, pH 7, is prevailing in the protein isolated from L. minor. The amino acid composition of L. minor protein in comparison with that of hen egg shows high contents of exogenous amino acids. The nutritional value of L. minor calculated on the basis of the amino acid composition is very high. The molecular weight of the protein fractions were estimated as above 176,000 and below 14,000.
This research is a continuation of our previous work on protein in Lemna minor et al. 1970). In the present investigation by control of growth conditions we tried to obtain a continous culture of Lemna minor producing the biomass of high biological value. We decided to determine the amino acid composition of the Lemna protein and to estabilish its biological value. The content of essential amino acids, namely: leucine, isoleucine, phenylalanine, lysine, methionine, threonine and tryptophan is a factor determining the nutritional value of protein. According to BLAIM (1964) the nutritional value of protein expressed as the so called EAA index (essential amino acid index) represents a mean value of the content of all essential amino acids as per cent of the standard protein that is of hen egg protein. As we found the protein soluble in orthophosphate buffer at pH 7 to be a predominant protein fraction we subjected it to gel filtration on Sephadex G-100 to determine a molecular weight of proteins composing this fraction. (MACIEJEWSKA-POTAPCZYK
Lemna minor was collected from its natural habitat (a pond) and then cultured in the laboratory on KNOP solution prepared with tap water and diluted to 1: 4. Light was provided by mercury lamps (2x400 Watt) at 16 light hours at temperature nearly 25 °C. Each 10-14 days Lemna minor was transfered to a freshly prepared nutrient solution. Under these conditions we observed a good growth of the Lemna biomass. The plants were harvested for analyses by filtering off. Samples were washed with distilled water, dried by a filter paper and weighed. The four-stage system of the protein extraction recommended by COATES and SIMMONDS (1961) is usually applied. To extract the Lemna proteins we used successively: 0.015 M orthophosphate buffer at pH 7, 0.05 M acetic acid and 0.1 M NaOH. The samples were shaken four times for 1 hour. We did not apply however any removal of lipids by water saturated with butanol- as in the original method - to avoid a transit of albumins and partially of globulins to the extract. At present the removal of lipids is usually omitted (JANKIEWICZ, POMERANZ 1965 a, b). Total protein was extracted
106
w. MACIEJEWSKA-POTAPCZYK, L.
KONOPSKA and K. OLECHNOWICZ
after FLETCHER and OSBORNE (1965). Total protein as well as protein of the respective fractions was determined by the LOWRY et al. method (1951). The molecular weight of protein extractable with 0.9 % NaCl in 0.01 M phosphate buffer pH 7 was determined in the way of gel filtration on Sephadex G-100. The Sephadex G-100 column was calibrated in the same way using the following standard proteins: lysozyme (Reanal), trypsin (2x cryst. salt free, Koch Light Laboratories LTD), pepsin (3 X cryst. B grade, Calbiochem), egg albumin (5 X cryst. reinst 99 %, Serva Entwicklungslabor Heidelberg), plasma albumin (Armour Pharmaceutical Company LTD), y-globulin fraction II (Koch Light Laboratories LTD). The protein fraction collected from the columns was determined spectrophotometrically at 280 nm and then verified by the LOWRY and coworkers (1951) method. 5 g samples of Lemna minor were used, acid-soluble and lipid fractions were removed and the remaining precipitate was dried under reduced pressure. Samples containing 5 mg of protein were subjected to the acid and alkaline hydrolysis according to KLYSZEJKO-STEFANOWICZ (1972). The amino acid composition of the acid hydrolyzate was determined by means of an automatic aminoacid analyzer (Carlo Erba 3A27 type). Tryptophan was determined in the alkaline hydrolyzate by the modified ECKERT'S procedure described by KLYSZEJKO-STEFANOWICZ (1972). All the above mentioned analyses were repeated at least 3 times.
Total protein in Lemna minor L. makes up 16 % of the dry matter. This content is v~ry high when compared with cereals in which it does not exceed 16 %. The same value was obtained in our previous research for Lemna minor cultivated under natural aquarium conditions that is together with other water plants and fish (MAClEJEWSKA-POTAPCZYK et al. 1970). The results of the respective protein fractions separation after COATES and SIMMONDS (1961) are shown in Table 1. The fraction clutable with 0.015 M orthophosphate buffer atpH 7 showed to be a prevailing one. Table 1. Protein fractions in Lemna minor L. Fractions
Protein in mg/100 mg of dry matter 1
2
3
average
13.10 13.37
14.81 14.80
14.20 14.57
14.14
Acetic acid 0.05M
0.35
0.31
0.27
0.31
0.1 M NaOH
1.57
1.71
1.60
1.63
Proteins (sum)l)
15.02 15.29
16.83 16.82
16.07 16.44
15.97
Protein (total)2)
15.18 15.64
16.87 16.10
18.31 17.00
16.50
Orthophosphate buffer 0.015 MpH 7
1) Calculated as the sum of respective fractions. 2) Total protein determined by the method of FLETCHER and OSBORNE.
Table 2 shows the amino acid content expressed as a percentage of dry matter and calculated for the total protein content. Low contents of tryptophan and sulphur amino acids and a relatively high content of the acidic amino acids should be emphasized.
Protein in Lemna minor L.
10'7
Table 2. Amino acid contents in Lemna minor L. protein Amino acid
mg/100 mg of dry matter mg/100 mg of protein
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Cystine Valine Methionine Izoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophanl)
1.67 0.86 0.68 0.82 2.28 1.24 1.33 traces 1.29 0.07 0.99 1.73 0.47 1.06 1.04 0.39 0.79 0.14
9.89 5.08 4.05 4.88 13.53 7.36 7.88 traces 7.67 0.39 5.89 10.27 2.76 6.28 6.20 2.32 4.67 0.85
1) Tryptophan was determined in alcaline hydrolysate as described in the methods. Table 3. Exogenous amino acids in Lemna minor L. total protein expressed in per cent and biologica value of Lemna as compared with that of egg protein Leu -leucine; lie - isoleucine; Val - valine; Phe - phenylalanine; Met - methionine; Lys lysine; Thr - threonine; Tyr - tyrosine. Material
Leu
lie
Val
Phe
Met
Lys
Thr
Lemna minor
10.27
7.67 7.5
6.28 5.7
0.39 4.0
5.08
2.76
76
11.7
5.89 8.2
6.20
Egg protein
7.8
4.9
1.5
97
Tyr
EAA index
Comparing the values obtained for Lemna minor protein with those for hen egg protein (Table 3) we found higher phenylalanine and tyrosine contents occuring in Lemna minor. The contents of leucine, valine, lysine and threonine were approximate in both kinds of protein. "EAA index" calculated according to OSER (cit. after MASLOWSKI1962): index EAA = 1/100aI • 100a 2 V bi b2
.••
100an bn
where: n - quantity of exogenous amino acids a - amino acids under examination b - amino acids in egg albumin shows that the nutritional value of Lemna calculated on the basis of the amino acid composition is very high.
108 W. MACIEJIlWSKA-POTAPCZYK, L. KONOPSKA and K. OLECHNOWICZ, Protein in Lemna minor L. The Lemna soluble protein was separated on Sephadex G-100 into 3 fractions: one of the high molecular weight above 176,000 and two low molecular fractions below 14,000. The high molecular fraction contributes to more than 50 % of soluble proteins. Lemna minor seems to be a substitute of high nutritional value. References BLAIM, K., Bialko roslinne jako zagadnienie biologiczne i rolnicze.
Post~py
Nauk Roln. 11, 47-52
(1964). COATES, J. H., SIMMONDS, D. H., Proteins of wheat and flour extraction, fractionation and chromatography of the buffer-soluble proteins of fluor. Cereal Chemistry 38, 256-272 (1961). FLETCLER, R. A., OSBORNE, D. J., Regulation of protein and nucleic acid synthesis by gibberellin during leaf senescence. Nature 207, 1176-1177 (1965). JANKIEWICZ, M., POMERANZ, Y., Isolation and characterisation of wheat flour proteins. I. Separation of salt and acetic acid-dispersible proteins by gel filtration polyacrylamide gel electrophoresis and sucrose gradient ultracentrifugation. J. Sci. Fd. Agric. 16, 644-652 (1965 a). - - Isolation and characterisation of wheat flour proteins. II. Effects of urea and N-ethylmaleimide on the behaviour of wheat proteins during extraction and fractionation. J. Sci. Fd. Agric. 16,
652-658 (1965 b). KLYSZEJKO-STEFANOWICZ, L., Cwiczenia z biochemii. PWN Warszawa 1972. LOWRY, J. O. H., ROSENBROUGH, N. J., FARR, A. L., RANDALL, R. J., Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-277 (1951). MACIEJEWSKA-POTAPCZYKOWA, W., KONOPSKA, L., NARZYMSKA, E., Proteins in duckweed (Lemna minor L.). Acta Soc. Bot. Pol. 39, 251-255 (1970). MASLOWSKI, P., Z badan nad bialkami kukurydzy. Aminokwasy egzogenne i wartosc biologiczna bialek r6znych odmian kukurydzy krajowej. Hodowla Roslin, Aklimatyzacja i Nasiennictwo
6, 605-613 (1962). Received August 2, 1974. Authors' address: Prof. Dr. W. MACIEJEWSKA-POTAPCZYK, L. KONOPSKA and K. OLECHNOWICZ, Department of Plant Physiology, Institute of Physiology and Cytology, University of Lodz, ul. Novopoludniowa 12/16, Lodz (Poland).
Short Communication
Protein in Lemna minor L. W.
MACIEJEWSKA-POTAPCZYK,
L.
KONOPSKA
and
K. OLECHNOWICZ
Department of Plant Physiology, Institute of Physiology and Cytology, University of Lodz, Lodz, Poland Key Term Index: bulk protein, amino acid composition, nutritional value; Lemna minor.
Summary By control of growth conditions a continuous culture of Lemna minor was obtained. Total protein in Lemna minor corresponds to 16 % of dry matter. The fraction elutable with 0.015 M orthophosphate buffer, pH 7, is prevailing in the protein isolated from L. minor. The amino acid composition of L. minor protein in comparison with that of hen egg shows high contents of exogenous amino acids. The nutritional value of L. minor calculated on the basis of the amino acid composition is very high. The molecular weight of the protein fractions were estimated as above 176,000 and below 14,000.
This research is a continuation of our previous work on protein in Lemna minor et al. 1970). In the present investigation by control of growth conditions we tried to obtain a continous culture of Lemna minor producing the biomass of high biological value. We decided to determine the amino acid composition of the Lemna protein and to estabilish its biological value. The content of essential amino acids, namely: leucine, isoleucine, phenylalanine, lysine, methionine, threonine and tryptophan is a factor determining the nutritional value of protein. According to BLAIM (1964) the nutritional value of protein expressed as the so called EAA index (essential amino acid index) represents a mean value of the content of all essential amino acids as per cent of the standard protein that is of hen egg protein. As we found the protein soluble in orthophosphate buffer at pH 7 to be a predominant protein fraction we subjected it to gel filtration on Sephadex G-100 to determine a molecular weight of proteins composing this fraction. (MACIEJEWSKA-POTAPCZYK
Lemna minor was collected from its natural habitat (a pond) and then cultured in the laboratory on KNOP solution prepared with tap water and diluted to 1: 4. Light was provided by mercury lamps (2x400 Watt) at 16 light hours at temperature nearly 25 °C. Each 10-14 days Lemna minor was transfered to a freshly prepared nutrient solution. Under these conditions we observed a good growth of the Lemna biomass. The plants were harvested for analyses by filtering off. Samples were washed with distilled water, dried by a filter paper and weighed. The four-stage system of the protein extraction recommended by COATES and SIMMONDS (1961) is usually applied. To extract the Lemna proteins we used successively: 0.015 M orthophosphate buffer at pH 7, 0.05 M acetic acid and 0.1 M NaOH. The samples were shaken four times for 1 hour. We did not apply however any removal of lipids by water saturated with butanol- as in the original method - to avoid a transit of albumins and partially of globulins to the extract. At present the removal of lipids is usually omitted (JANKIEWICZ, POMERANZ 1965 a, b). Total protein was extracted
106
w. MACIEJEWSKA-POTAPCZYK, L.
KONOPSKA and K. OLECHNOWICZ
after FLETCHER and OSBORNE (1965). Total protein as well as protein of the respective fractions was determined by the LOWRY et al. method (1951). The molecular weight of protein extractable with 0.9 % NaCl in 0.01 M phosphate buffer pH 7 was determined in the way of gel filtration on Sephadex G-100. The Sephadex G-100 column was calibrated in the same way using the following standard proteins: lysozyme (Reanal), trypsin (2x cryst. salt free, Koch Light Laboratories LTD), pepsin (3 X cryst. B grade, Calbiochem), egg albumin (5 X cryst. reinst 99 %, Serva Entwicklungslabor Heidelberg), plasma albumin (Armour Pharmaceutical Company LTD), y-globulin fraction II (Koch Light Laboratories LTD). The protein fraction collected from the columns was determined spectrophotometrically at 280 nm and then verified by the LOWRY and coworkers (1951) method. 5 g samples of Lemna minor were used, acid-soluble and lipid fractions were removed and the remaining precipitate was dried under reduced pressure. Samples containing 5 mg of protein were subjected to the acid and alkaline hydrolysis according to KLYSZEJKO-STEFANOWICZ (1972). The amino acid composition of the acid hydrolyzate was determined by means of an automatic aminoacid analyzer (Carlo Erba 3A27 type). Tryptophan was determined in the alkaline hydrolyzate by the modified ECKERT'S procedure described by KLYSZEJKO-STEFANOWICZ (1972). All the above mentioned analyses were repeated at least 3 times.
Total protein in Lemna minor L. makes up 16 % of the dry matter. This content is v~ry high when compared with cereals in which it does not exceed 16 %. The same value was obtained in our previous research for Lemna minor cultivated under natural aquarium conditions that is together with other water plants and fish (MAClEJEWSKA-POTAPCZYK et al. 1970). The results of the respective protein fractions separation after COATES and SIMMONDS (1961) are shown in Table 1. The fraction clutable with 0.015 M orthophosphate buffer atpH 7 showed to be a prevailing one. Table 1. Protein fractions in Lemna minor L. Fractions
Protein in mg/100 mg of dry matter 1
2
3
average
13.10 13.37
14.81 14.80
14.20 14.57
14.14
Acetic acid 0.05M
0.35
0.31
0.27
0.31
0.1 M NaOH
1.57
1.71
1.60
1.63
Proteins (sum)l)
15.02 15.29
16.83 16.82
16.07 16.44
15.97
Protein (total)2)
15.18 15.64
16.87 16.10
18.31 17.00
16.50
Orthophosphate buffer 0.015 MpH 7
1) Calculated as the sum of respective fractions. 2) Total protein determined by the method of FLETCHER and OSBORNE.
Table 2 shows the amino acid content expressed as a percentage of dry matter and calculated for the total protein content. Low contents of tryptophan and sulphur amino acids and a relatively high content of the acidic amino acids should be emphasized.
Protein in Lemna minor L.
10'7
Table 2. Amino acid contents in Lemna minor L. protein Amino acid
mg/100 mg of dry matter mg/100 mg of protein
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Cystine Valine Methionine Izoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophanl)
1.67 0.86 0.68 0.82 2.28 1.24 1.33 traces 1.29 0.07 0.99 1.73 0.47 1.06 1.04 0.39 0.79 0.14
9.89 5.08 4.05 4.88 13.53 7.36 7.88 traces 7.67 0.39 5.89 10.27 2.76 6.28 6.20 2.32 4.67 0.85
1) Tryptophan was determined in alcaline hydrolysate as described in the methods. Table 3. Exogenous amino acids in Lemna minor L. total protein expressed in per cent and biologica value of Lemna as compared with that of egg protein Leu -leucine; lie - isoleucine; Val - valine; Phe - phenylalanine; Met - methionine; Lys lysine; Thr - threonine; Tyr - tyrosine. Material
Leu
lie
Val
Phe
Met
Lys
Thr
Lemna minor
10.27
7.67 7.5
6.28 5.7
0.39 4.0
5.08
2.76
76
11.7
5.89 8.2
6.20
Egg protein
7.8
4.9
1.5
97
Tyr
EAA index
Comparing the values obtained for Lemna minor protein with those for hen egg protein (Table 3) we found higher phenylalanine and tyrosine contents occuring in Lemna minor. The contents of leucine, valine, lysine and threonine were approximate in both kinds of protein. "EAA index" calculated according to OSER (cit. after MASLOWSKI1962): index EAA = 1/100aI • 100a 2 V bi b2
.••
100an bn
where: n - quantity of exogenous amino acids a - amino acids under examination b - amino acids in egg albumin shows that the nutritional value of Lemna calculated on the basis of the amino acid composition is very high.
108 W. MACIEJIlWSKA-POTAPCZYK, L. KONOPSKA and K. OLECHNOWICZ, Protein in Lemna minor L. The Lemna soluble protein was separated on Sephadex G-100 into 3 fractions: one of the high molecular weight above 176,000 and two low molecular fractions below 14,000. The high molecular fraction contributes to more than 50 % of soluble proteins. Lemna minor seems to be a substitute of high nutritional value. References BLAIM, K., Bialko roslinne jako zagadnienie biologiczne i rolnicze.
Post~py
Nauk Roln. 11, 47-52
(1964). COATES, J. H., SIMMONDS, D. H., Proteins of wheat and flour extraction, fractionation and chromatography of the buffer-soluble proteins of fluor. Cereal Chemistry 38, 256-272 (1961). FLETCLER, R. A., OSBORNE, D. J., Regulation of protein and nucleic acid synthesis by gibberellin during leaf senescence. Nature 207, 1176-1177 (1965). JANKIEWICZ, M., POMERANZ, Y., Isolation and characterisation of wheat flour proteins. I. Separation of salt and acetic acid-dispersible proteins by gel filtration polyacrylamide gel electrophoresis and sucrose gradient ultracentrifugation. J. Sci. Fd. Agric. 16, 644-652 (1965 a). - - Isolation and characterisation of wheat flour proteins. II. Effects of urea and N-ethylmaleimide on the behaviour of wheat proteins during extraction and fractionation. J. Sci. Fd. Agric. 16,
652-658 (1965 b). KLYSZEJKO-STEFANOWICZ, L., Cwiczenia z biochemii. PWN Warszawa 1972. LOWRY, J. O. H., ROSENBROUGH, N. J., FARR, A. L., RANDALL, R. J., Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-277 (1951). MACIEJEWSKA-POTAPCZYKOWA, W., KONOPSKA, L., NARZYMSKA, E., Proteins in duckweed (Lemna minor L.). Acta Soc. Bot. Pol. 39, 251-255 (1970). MASLOWSKI, P., Z badan nad bialkami kukurydzy. Aminokwasy egzogenne i wartosc biologiczna bialek r6znych odmian kukurydzy krajowej. Hodowla Roslin, Aklimatyzacja i Nasiennictwo
6, 605-613 (1962). Received August 2, 1974. Authors' address: Prof. Dr. W. MACIEJEWSKA-POTAPCZYK, L. KONOPSKA and K. OLECHNOWICZ, Department of Plant Physiology, Institute of Physiology and Cytology, University of Lodz, ul. Novopoludniowa 12/16, Lodz (Poland).