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Determination of Soil Enzyme Activity and Extraction, Purification and MS Identification of Soil Microbial Total Protein

Author: WuYun
Tutor: CaoHui
School: Nanjing Agricultural College
Course: Microbiology
Keywords: 2D-PAGE MS Proteome Peptide Mass Fingerprinting(PMF) Density Gradient Centrifugation the activity of soil enzyme
CLC: S154
Type: Master's thesis
Year: 2011
Downloads: 64
Quote: 0
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Abstract


Soil enzyme is a component of the soil. Soil enzyme activities are closely related to soil physical properties, water and heat conditions, the soil organic and inorganic components of complex chemical composition and the characteristics of absorption. Metaproteomics is a new research field on microbial community based on proteomics, the aim of which is to give an instant and broad scale analysis on a mixed of environmental microorganisms in a given space by its definition. Though it was still at a primary stage, studies on metaproteomics already show its power in fields of microorganisms in a given biotope. The aim of our work was to measure soil enzyme activities under different fertilization, we extract, purify soil bacteria using density gradient centrifugation. For cell protein using sonication, then ultrafiltration and TCA precipitation to purify cell protein. By SDS-PAGE to seprate cell protein. Further purified bacterial protein with 2D-PAGE technique, obtaining electrophoresis map of better quality. To analyze soil cell proteins commonality and differences under three different treatments. We identify soi lmicrobial proteins using Matrix-assisted laser desorption/Ionization time of flight mass spectrometry （MALDI-TOF-MS）, analyze the presence problems of soil metaproteomics.In three treatment conditions（CK, NPK, M）, the biomass,physical and chemical properties of reddish paddy soil are all changed. Proved fertilization in different ways, soil enzyme activity differences also exist. Soil urease activity of CK. is 0.074 mg NH3-N g-1 soil·3h-1, NPK is 0.108 mg NH3-N·g-1 soil·3h-1, and M in the soil urease activity is 0.191 mg NH3-N·g-1 soil·3h-1; protease activity is 2.78μg Tyr·g-1 soil·48h-1,7.55μg Tyr·g-1 soil·48h-1,8.22μg Tyr·g-1 soil·48h-1; invertase activity is 1.06 mg glucose. g-1 soil,1.08 mg glucose·g-1soil,1.35 mg glucose·g-1 soil. The effect of NPK on urease and protease is larger, and less effect on the invertase activity, M effect on soil enzyme is protease＞urease＞invertase. Determination of soil microbial enzyme activity on the show, intracellular is larger than extracelluar. Extracelluar urease activity is CK<NPK<CK+sterilized soil<M<NPK+sterilized soil<M+sterilized soil. To the same soil sample, soil urease activity is significantly increased after adding sterile soil. To different soil amples, M has a larger increase than NPK.Intracelluar urease activity is CK<CK+sterilized soil<NPK<NPK+sterilized soil<M<M+sterilized soil. As the intracelluar, to the same soil sample,soil urease activity is significantly increased after adding st erile soil, to different soil amples, M has a larger increase than NPK.We count primary soil and extracted cells from soils by DAPI staining and plate counting, the counting result showed that DAPI counting are two orders of magnitude larger than plate counting. The soil microbial protein content is very low by Density gradient centrifugation, the soil CK is 0.4μg·g-1 soil, NPK is 0.68μg·g-1 soil and 0.78μg·g-1soil. We can find that three soil samples of the protein have differences apparently by SDS-PAGE.Proteins are mainly concentrated between the 44.32 Kda and 92.7 Kda.Becauce the sample protein extraction, there are still some humic acid impurities are difficult to remove. We have to choose low salt concentration buffer to purify protein to obtain better map in 2D-PAGE, and we should centrifugal in 4℃,12000 r/min for 1h. We get three commen protein and eight different protein from three soil samples by Matrix-assisted laser desorption/Ionization time of flight mass spectrometry （MALDI-TOF-MS）. Three common proteins are Hypothetical protein FAEPRAM21202949; phage prohead protease, HK97 family; Glutamate synthase, NADH/NADPH, small subunit;eight different proteins are recombinase A; transcriptional regulator, GntR family; Conserved hypothetical protein; AFG1-like ATPase; 3-hydroxy-3-methylglutaryl-CoA reductase; outer membrane protein A; putative ATP-dependent helicase; Mg chelatase-related protein.

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CLC: > Agricultural Sciences > Agriculture as the foundation of science > Soil > Soil Biology
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