Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2000 Arylamidase activity of soils Verónica Acosta-Martínez Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, and the Soil Science Commons Recommended Citation Acosta-Martínez, Verónica, "Arylamidase activity of soils " (2000). Retrospective Theses and Dissertations. 12500. https://lib.dr.iastate.edu/rtd/12500 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. 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Higher quality 6' x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additk)nal charge. Contact UMI directly to order. Bell & Howell Informatton and Learning 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 800-521-0600 Arylamidase activity of soils by Ver6nica Acosta-Martinez A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Soil Science (Soil Microbiology and Biochemistry) Major Professor: M. A. Tabatabai Iowa State University Ames, Iowa 2000 UMI Number 9962791 UMI^ UMI Microfomfi9962791 Copyright 2000 by Bell & Howell Infomiatlon and Learning Company. All rights reserved. This microfomi edKion is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the Doctoral dissertation of Veronica Acosta-Martinez has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major Professor Signature was redacted for privacy. For the Major Program Signature was redacted for privacy. •or the;6waduate College m DEDICATION To my family, e^edally my son Sergio Jose iv TABLE OF CONTENTS Page INTRODUCTION I LITERATURE REVIEW 4 Soil Enzymes 4 Amidohydiolases 7 Glycosidases 10 Phosphatases and Arysulfatase 11 Nitrogen Mineralization 14 Arylamidase 19 Soil Quality 24 Liming Effect on Soil Quality 26 Tillage and Residue Management Effects on Enzyme Activities in Soil 29 PART L ASSAY OF ARYLAMIDASE ACTIVITY IN SOILS 32 INTRODUCTION 33 MATERIALS AND METHODS 35 Soils and their Properties 35 Reagents 35 Assay Procedure 38 Experiments 41 V Page RESULTS AND DISCUSSION 43 Buffer pH 43 Substrate Concentration and Amount of Soil 45 Procedure to Terminate the Reaction after Incubation 45 Time and Temperature of Incubation 48 Activation Energy, Enthalpy of Activation, and Kinetic Parameters 54 Effects of Various Treatments 63 Precision of the Method 66 PART II. ARYLAMIDASE ACTIVITY OF SOILS: EFFECT OF TRACE ELEMENTS AND RELATIONSHIPS TO ACTIVITIES OF AMIDOHYDROLASES 68 INTRODUCTION 69 MATERIALS AND METHODS 72 Soils 72 Trace Elements 72 Assay of Arylamidase Activity 75 RESULTS AND DISCUSSION 76 Effect of Trace Elements 76 Effect of Soil Properties 80 Relationship between Arylamidase Activity and Activities of Amidohydrolases 85 vi Page PART m. EFFECT OF TOLUENE ON ARYLAMTOASE ACTIVITY IN SOILS 88 INTRODUCTION 89 MATERIAL AND METHODS 92 Soils and their Properties 92 Reagents 92 Experiments 92 Assay of Arylamidase Activity 94 RESULTS AND DISCUSSION 96 Buffer pH 96 Substrate Concentration and Amount of SoU 98 Time and Temperature of Incubation 100 Activation Energy, Enthalpy of Activation and Kinetic Parameters 104 PART IV. SUBSTRATE SPECIFICITY OF ARYLAMTOASE IN SOILS 117 INTRODUCTION 118 MATERIALS AND METHODS 119 Soils and their Properties 119 Reagents 119 Assay of Arylamidase Activity 119 vii Page RESULTS AND DISCUSSION 123 Buffer pH 123 Substrate Specificity 126 Kinetic Parameters 126 Activation Energy, Qio, and Enthalpy of Activation 130 PART V. ENZYME ACTIVITIES IN A LIMED AGRICULTURAL SOIL 137 INTRODUCTION 138 MATERIALS AND METHODS 140 Experimental Design 140 Sampling and Laboratory Analyses 140 RESULTS AND DISCUSSION 144 Activities of Glycosidases 144 Activities of Amidohydrolases and Arylamidase 150 Activities of Phosphatases and Arylsulfatase 154 PART VI. EFFECT OF TILLAGE AND RESIDUE MANAGEMENT ON ARYLAMIDASE ACTIVITY IN SOILS 159 INTRODUCTION 160 viii Page MATERIALS AND METHODS 163 Treatments Description 163 Soil Sampling and Laboratory Analyses 164 Assay of Arylamidase Activity 164 Statistical Analysis 167 RESULTS AND DISCUSSION 168 SUMMARY AND CONCLUSIONS 175 APPENDIX 180 LITERATURE CITED 182 ACKNOWLEDGEMENTS 205 1 INTRODUCTION Soil, as a living system contains many enzymes. Most soil enzymes are primarily of microbial origin, but may also originate firom animal and plant residues (Kiss et al., 1975; Bums, 1982). Enzjrmes are present in soils inside (intracellular) living cells, in the periplasmic space and outer cell surfaces stabilized on the clay-organic matter complexes (Bums, 1982). Soil enzymes play a key role in the decomposition of various organic compounds, and in the decomposition and the formation of the soil organic matter. Thus, enzymes are important components involved in the dynamics of the cycling of the nutrients C, N, P, and S in soils. Among the most important nutrients in soils, N is the most needed by most agricultural crops but only trace quantities are available in the mineral form. The organic form is a major component of soil organic matter and may account for greater than 95% of the total N in most surface soils. About half of this organic N has not yet been identified. It has been estimated that about 20- 40 % of the total N in soils is present in the form of amino adds, but only a small portion of the amino adds is present in a "free" state and the major portion is bound to soil organic matter (Bremner, 1951). Several researchers (Grov, 1963; Grov and Alvsaker, 1963; Ivarson andSowden, 1966, 1970; Paul and Schmidt, 1960; Stevenson, 1982, 1985; Monreal and McGill, 1985) have studied the portion of "firee" amino adds in soils because it is an available energy source for 2 soil microorganisms (Ivarson and Sowden, 1966) and an available N source for plant growth (Broadbent, 1984). The amino acids bound to the soil organic matter are most likely in the form of proteins or associated to the clay-organic matter complexes as peptides, amides or arylamides. These amino acids are released from the soil organic matter by the activities of enzsrmes such as arylamidase, and later the "free" amino acids are hydrolyzed by specific enzymes, producing NH4'^, which, in turn, is nitrified for plant uptake. Among the amidohydrolases, L-aspartase, L-asparaginase, and L- glutaminase are enzjones acting on "free" amino adds of soils. The amidohydrolases are very specific in that they act on the C-N bonds other than peptide bonds in linear amides releasing NH4'*'. Many researchers have reported about the activities of such amidohydrolases and the factors that affect their activities (Frankenberger and Tabatabai, 1991c,d; Tabatabai, 1994; Senwo and Tabatabai, 1996,1999). The enzyme amino add arylamidase [a-aminoacyl-peptide hydrolase (microsomal) EC 3.4.11.2] catalyzes the hydrolysis of an N-terminal amino add from peptides, amides, or arylamides. This enzyme has been detected in the tissues and body fluids of all animals (Hiwada et al., 1980), plants, and microorganisms (Appel, 1974). Arylamidase may play a key role in the initial reactions of the mineralization of amino adds in soils but no information is available about this enzyme in soils. Studies to understand the role of arylamidase in soil N cycling and the factors (i.e., soil properties, trace elements, 3 liming, tillage and crop residues management practices) that affect the activity of this enzyme will aid in decision making important for the fertility, productivity and sustainabiUty of soils. The objectives of this work were: (1) to develop a method to assay the activity of arylamidase in soils, (2) to assess the factors affecting arylamidase activity in soils and its relationship to the amidohydrolases, (3) to assess the effect of trace elements on the activity of this enzyme in soils, (4) to determine the effect of toluene on the activity of arylamidase, (5) to study the substrate specificity of arylamidase, (6) to evaluate the effect of liming on the activities of 14 different enzymes, including arylamidase, and (7) to evaluate the influence of tillage and residue management on the activity of arylamidase. The resiilts obtained are presented in six parts. The results obtained under objective (1) are presented in Part I, those under objective (2 and 3) are presented in Part II, those under objective (4) are presented in Part m, those under objective (5) are presented in Part IV, those under objective (6) are presented in Part V, and those imder objective (7) are presented in Part VI. Preceding Part I is a literatiire review and after Part VI is a summary and conclusions.