Understanding immobilised enzymes by NMR spectroscopy

Enzyme immobilisation is the conversion of a soluble enzyme molecule into a solid particle form. This allows the recovery of the enzyme catalyst for its re-use and avoids protein contamination of the product streams. A better understanding of immobilised enzymes is necessary for their rational development. A more rational design can help enormously in the applicability of these systems in different areas, from biosensors to chemical industry. Immobilised enzymes are challenging systems to study and very little information is given by conventional biochemical analysis such as catalytic activity and amount of protein. Here, solid-state NMR has been applied as the main technique to study these systems and evaluate them more precisely. Various approaches are presented for a better understanding of immobilised enzymes, which is the aim of this thesis. Firstly, the requirements of a model system of study will be discussed. The selected systems will be comprehensibly characterised by a variety of techniques but mainly by solid-state NMR. The chosen system will essentially be the enzyme α-chymotrypsin covalently immobilised on two functionalised inorganic supports – epoxide silica and epoxide alumina – and an organic support – Eupergit®. The study of interactions of immobilised enzymes with other species is vital for understanding the macromolecular function and for predicting and engineering protein behaviour. The study of water, ions and inhibitors interacting with various immobilised enzyme systems is covered here. The interactions of water and sodium ions were studied by 17O and 23Na multiple-quantum techniques, respectively. Various pore sizes of the supports were studied for the immobilised enzyme in the presence of labelled water and sodium cations. Finally, interactions between two fluorinated inhibitors and the active site of the enzyme will be explored using 19F NMR, offering a unique approach to evaluate catalytic behaviour. These interactions will be explored by solution-state NMR firstly, then by solid-state NMR. NMR has the potential to give information about the state of the protein in the solid support, but the precise molecular interpretation is a difficult task.

A SUBSET OF 3´ TO 5´ EXORIBONUCLEASES ARE THE PRIMARY ENZYMES RESPONSIBLE FOR THE DEGRADATION OF PGPG IN CYCLIC DI-GMP SIGNALING

Bis-(3´-5´)-cyclic dimeric guanosine monophosphate, or cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that regulates processes such biofilm formation, motility, and virulence. C-di-GMP is synthesized by diguanylate cyclases (DGCs), while phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMPs by previously unidentified enzymes termed PDE-Bs. To identify the PDE-B responsible for pGpG turnover, a screen for pGpG binding proteins in a Vibrio cholerae open reading frame library was conducted to identify potential pGpG binding proteins. This screen led to identification of oligoribonuclease (Orn). Purified Orn binds to pGpG and can cleave pGpG to GMP in vitro. A deletion mutant of orn in Pseudomonas aeruginosa was highly defective in pGpG turnover and accumulated pGpG. Deletion of orn also resulted in accumulation c-di-GMP, likely through pGpG-mediated inhibition of the PDE-As, causing an increase in c-di-GMP-governed auto-aggregation and biofilm. Thus, we found that Orn serves as the primary PDE-B enzyme in P. aeruginosa that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway. However, not all bacteria that utilize c-di-GMP signaling also have an ortholog of orn, suggesting that other PDE-Bs must be present. Therefore, we asked whether RNases that cleave small oligoribonucleotides in other species could also act as PDE-Bs. NrnA, NrnB, and NrnC can rapidly degrade pGpG to GMP. Furthermore, they can reduce the elevated aggregation and biofilm formation in P. aeruginosa ∆orn. Together, these results indicate that rather than having a single dedicated PDE-B, different bacteria utilize distinct RNases to cleave pGpG and complete c-di-GMP signaling. The ∆orn strain also has a growth defect, indicating changes in other regulatory processes that could be due to pGpG accumulation, c-di-GMP accumulation, or another effect due to loss of Orn. We sought to investigate the genetic pathways responsible for these growth defect phenotypes by use of a transposon suppressor screen, and also investigated transcriptional changes using RNA-Seq. This work identifies that c-di-GMP degradation intersects with RNA degradation at the point of the Orn and the functionally related RNases.

Study of tRNA modifying enzymes and codon usage bias in cancer

Recent evidences indicate that tRNA modifications and tRNA modifying enzymes may play important roles in complex human diseases such as cancer, neurological disorders and mitochondrial-linked diseases. We postulate that expression deregulation of tRNA modifying enzymes affects the level of tRNA modifications and, consequently, their function and the translation efficiency of their tRNA corresponding codons. Due to the degeneracy of the genetic code, most amino acids are encoded by two to six synonymous codons. This degeneracy and the biased usage of synonymous codons cause alterations that can span from protein folding to enhanced translation efficiency of a select gene group. In this work, we focused on cancer and performed a meta-analysis study to compare microarray gene expression profiles, reported by previous studies and evaluate the codon usage of different types of cancer where tRNA modifying enzymes were found de-regulated. A total of 36 different tRNA modifying enzymes were found de-regulated in most cancer datasets analyzed. The codon usage analysis revealed a preference for codons ending in AU for the up-regulated genes, while the down-regulated genes show a preference for GC ending codons. Furthermore, a PCA biplot analysis showed this same tendency. We also analyzed the codon usage of the datasets where the CTU2 tRNA modifying enzyme was found deregulated as this enzyme affects the wobble position (position 34) of specific tRNAs. Our data points to a distinct codon usage pattern between up and downregulated genes in cancer, which might be caused by the deregulation of specific tRNA modifying enzymes. This codon usage bias may augment the transcription and translation efficiency of some genes that otherwise, in a normal situation, would be translated less efficiently.

Understanding the role of auxins and oxidative enzymes on adventitious root formation in olive (olea europaea L.) cultivars

Olive (Olea europaea L.), one of the main crops in the Mediterranean basin, is mainly propagated by cuttings, a classical propagation method that relies on the ability of the cuttings to form adventitious roots. While some cultivars are easily propagated by this technique, some of the most interesting olive cultivars are considered difficult-to-root which poses a challenge for their preservation and commercialization. Therefore, increasing the current knowledge on adventitious root formation is extremely important for species like olive. This research focuses on evaluating the role of free auxins and oxidative enzymes on adventitious root formation of two olive cultivars with different rooting ability - ‘Galega vulgar’ (difficult-to-root) and ‘Cobrançosa’ (easy-to-root). In this context, free auxin levels and enzyme activities were determined in in vitro-cultured ‘Galega vulgar’ microshoots and in semi-hardwood cuttings of cvs. ‘Galega vulgar’ and ‘Cobrançosa’. To attain this goal, an analytical method for the quantification of free indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) was developed, which is based on dispersive liquid-liquid microextraction followed by microwave derivatization (DLLME-MAD) and gas chromatography-mass spectrometry (GC/MS) analysis. The developed method was validated in terms of linearity, recovery, limit of detection (LOD) and limit of quantification (LOQ) and proved to be useful in the analysis of two very different types of plant tissues. The results from auxin quantification in olive samples point at a relationship between free auxin levels and rooting ability of both microshoots and semihardwood cuttings. A defective IBA-IAA conversion, resulting in a peak of free IAA during initiation phase, seems to be associated with low rooting ability. Likewise, differences in the activity of oxidative enzymes also appear to be related with rooting ability. Higher polyphenol oxidases (PPO) activity is likely related with an easyto- root behavior, while the opposite is true for peroxidases (POX) (including IAA oxidase (IAAox)) activity. A possible hypothesis for adventitious root formation in olive microcuttings is presented herein for the first time. Free auxins, oxidative enzymes, alternative oxidase (AOX) and reactive oxygen species (ROS) are some of the factors that may be involved in this highly complex physiological process. Interestingly, while temporal changes in auxin levels were similar between microshoots and semihardwood cuttings, the conclusions obtained from enzyme activity results in microshoots didn’t translate to semi-hardwood tissues, showing the emerging need for adaptation of classical agronomical research studies to modern techniques; Resumo: Procurando compreender o papel das auxinas e enzimas oxidativas na formação de raízes adventícias em cultivares de oliveira (Olea europaea L.) A oliveira (Olea europaea L.) é uma das principais culturas da bacia Mediterrânica e é propagada maioritariamente por estacaria, um processo altamente dependente da capacidade das estacas para formar raízes adventícias. Enquanto algumas cultivares são fáceis de propagar desta forma, algumas das cultivares de oliveira mais interessantes são consideradas difíceis de enraizar, o que dificulta a sua preservação e comercialização e torna extremamente importante aprofundar o conhecimento sobre o enraizamento adventício desta espécie. Este trabalho foca-se na avaliação do papel das auxinas livres e das enzimas oxidativas na formação de raízes adventícias em duas cultivares de oliveira com diferente capacidade de enraizamento - ‘Galega vulgar’ (difícil de enraizar) e ‘Cobrançosa’ (fácil de enraizar). Neste contexto, determinaram-se os níveis de auxinas livres e as actividades de enzimas oxidativas em microestacas de ‘Galega vulgar’ cultivadas in vitro bem como em estacas semi-lenhosas das cvs. ‘Galega vulgar’ e ‘Cobrançosa’. Para tal foi necessário desenvolver uma metodologia analítica para a quantificação de ácido indol-3-acético (IAA) e ácido indol-3-butírico (IBA), baseada em microextracção dispersiva líquido-líquido (DLLME) seguida de derivatização em microondas (MAD) e análise por cromatografia gasosa acoplada a espectrometria de massa (GC/MS). O método desenvolvido foi validado em termos de linearidade, recuperação, limite de detecção (LOD) e limite de quantificação (LOQ), e mostrou-se eficaz na análise de dois tipos de tecidos vegetais bastante diferentes. Os resultados da análise de auxinas em amostras de oliveira apontam para uma possível relação entre os níveis de auxinas livres e a capacidade de enraizamento, tanto em microestacas como em estacas semi-lenhosas. Uma conversão IBA-IAA deficiente, que resulta num pico de IAA durante a fase de iniciação, parece estar associada à baixa capacidade de enraizamento. Por outro lado, a capacidade de enraizamento também parece estar relacionada com diferenças na actividade de enzimas oxidativas. Comportamentos fáceis de enraizar estão associados a actividade mais elevada das polifenoloxidases (PPO), enquanto o oposto é verdade para a actividade das peroxidases (POX) (incluindo a IAA oxidase (IAAox)). Neste trabalho propõe-se pela primeira vez uma possível explicação para o enraizamento adventício em microestacas de oliveira. Auxinas livres, enzimas oxidativas, oxidase alternativa (AOX) e espécies reactivas de oxigénio (ROS) são alguns dos factores envolvidos neste processo fisiológico altamente complexo. Curiosamente, enquanto as alterações temporais nos níveis de auxinas foram semelhantes entre microestacas e estacas semi-lenhosas, o mesmo não se observou relativamente à actividade enzimática, o que mostra a necessidade de adaptação dos estudos agronómicos tradicionais às técnicas correntes.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

ENZYME-FREE UBIQUITIN LIGATION. FROM NATIVE CHEMICAL LIGATION AND SPIN LABELING TO DIMERIZATION BY INTER-UBIQUITIN MIMICKING LINKAGE AND PH-DEPENDENT CONFORMATIONAL SWITCH

The delicate balance between the production and disposal of proteins is vital for the changes required in the cell to respond to given stimulus. Ubiquitination is a protein modification with a range of signaling outcomes when ubiquitin is attached to a protein through a highly ordered enzymatic cascade process. Understanding ubiquitination is a growing field and nowadays the application of chemical reactions allows the isolation of quantitative materials for structural studies. Therefore, in this dissertation it is described some of these suitable chemical methodologies to produce an isopeptide bond toward the polymerization of ubiquitin bypassing the enzymatic control with the purpose of showing if these chemical modifications have a direct impact on the structure of ubiquitin. First, the possibility of incorporating non-natural lysine analogs known as mercaptolysines into the polypeptide chain of Ubiquitin was explored when they were attached to ubiquitin by native chemical ligation at its C terminus. The sulfhydryl group was used for the attachment of a paramagnetic label to map the surface of ubiquitin. Second, the condensation catalyzed by silver nitrate was used for the dimer assembly. In particular, the main focus was on examining whether orthogonal protection and deprotection of each monomer have an impact on the reaction yield, since the synthetic strategy has been previously attempted successfully. Third, the formation of ubiquitin dimers was approached by building an inter-ubiquitin linkage mimicking the isopeptide bond with two approaches, the classic disulfide exchange as well as the thiol-ene click reaction by thermal initiation in aqueous conditions. After assembling the dimeric units, they were studied by Nuclear Magnetic Resonance, in order to establish a conformational state profile which depends on the pH conditions. The latter is a very important concept since some ligands have a preferred affinity when the protein-protein hydrophobic patches are in close proximity.

Optimization of Enzymes Inactivation in High Pressure Processes

The demand of highest quality foods in terms of taste and their properties preservation without the use of additives is constantly increasing. Consequently, new approaches to food processing have been developed, as for example high-pressure technology which has proven to be very valuable because it allows to maintain good properties of food like some vitamins and, at the same time, to reduce some undesirable bacteria. This technology avoids the use of high temperatures during the process (not like Pasteurization), which may have adverse effect on some nutritional properties of the food, its flavour, etc. The models for some enzymatic inactivations, which depend on the pressure and temperature profiles are presented. This work deals with the optimization of the inactivation of certain enzymes when high pressure treatment on food processing is applied. The optimization algorithms will minimize the inactivation not only of a certain isolated enzyme but also to several enzymes that can be involved simultaneously in the high-pressure process.

Influence of enzymes and technology on the composition of Cobrançosa and Galega vulgar virgin olive oils

Doutoramento em Engenharia Alimentar - Instituto Superior de Agronomia - UL

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

There has been some concern about the environmental impact of microbial agents. Pseudomonas may be used as bioremediator and as biopesticide. In this study, we report the use of soil enzyme assays as biological indicator of possible negative effects in soil functioning after the P. putida AF7 inoculation. For that, P. putida AF7 was originally isolated from the rizosphere of rice and was inoculated on three soil types: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). The acid phosphatase, b-glucosidase and protease enzymes activities were measured for three period of evaluation (7, 14 and 21 days). In general, the enzymatic activities pre- sented variation among the tested soils. The highest activities of b-glucosidase and acid phosphatase were observed in the RH and AH soils, while the protease activity was higher in the TH soil. Also, the soil charac- teristics were measured for each plot. The activity of enzymes from the carbon cycle was positively correlated with the N and the P and the enzyme from the nitrogen cycle was negatively correlated with N and C.org. The presented data indicate that soil biochemical properties can be an useful tool for use as an indicator of soil perturba- tions by microbial inoculation in a risk assessment.

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.

Importance of the Support Properties for Immobilization or Purification of Enzymes

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed.

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ2235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC50 on ATP concentration that allows prediction of the IC50 at different ATP concentrations in enzyme and cellular assays. Comparison of the drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K/PTEN/AKT/mTOR1 network in order to understand mechanisms of drug sensitivity and resistance in different cancer cell lines. We suggest that using these models in systems biology investigation of the PI3K/AKT/mTOR1 signalling in cancer cells can bridge the gap between direct drug target action and the therapeutic response to these drugs and their combinations.

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

Pseudomonas may use as bioremediator and as biopesticide. The use of soil enzymatic assays as biological indicator of possible negative effects in soil functioning was evaluated after P.putida AF7 inoculation. For that, AF7 was originally isolated from the rizosphere of rice and was inoculated on three soils: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). Soil characteristics were measured in each plot. Acid phosphatase, ?-glucosidase and protease activities were measured at 7, 14 and 21 days. The enzyme activity waved during the experimental period but there is a significant reduction of ?-glucosidase activity in RH soil on day 14. Corg was positively correlated to the activities of ?-glucosidase and protease. The presented data indicate that soil biochemical properties may be useful as indicator of soil perturbations.

Influence of pseudomonas putida af7 inoculation on soil enzymes activities.

2008