Isolation and biochemical characterization of novel acid phosphatase and zinc-dependent acid phosphatase from the chicken's brain.

The AcPase exhibits a specific activity of 31.32 U/mg of protein with a 728-fold purification, and the yield of the enzyme is raised to 3.15 %. The Zn2+-dependent AcPase showed a purification factor of 1.34 specific activity of 14 U/mg of proteins and a total recovery of 5.14. The SDS-PAGE showed a single band corresponding to a molecular weight of 18 kDa of AcPase and 29 kDa of Zn2+-dependent AcPase. The AcPase enzyme has shown a wide range of substrate specificity for p-NPP, phenyl phosphate and FMN, while in the case of ZnAcPase α and ß-Naphthyl phosphate and p-NPP were proved to be superior substrates. The divalent metal ions like Mg2+, Mn2+, and Ca2+ increased the activity, while other substrates decreased the enzyme activity. The Km (0.14 mM) and Vmax (21 µmol/min/mg) values of AcPase were higher than those of Zn2+-AcPase (Km = 0.5 mM; Vmax = 9.7 µmol/min/mg). The Zn2+ ions activate the Zn2+-AcPase while Fe3+, Al3+, Pb2+, and Hg2+ showed inhibition on enzyme activity. Molybdate, vanadate and phosphate were found to be competitive inhibitors of AcPase with Ki values 316 µM, 185 µM, and 1.6 mM, while in Zn2+-AcPase tartrate and phosphate also showed competitive inhibition with Ki values 3 mM and 0.5 mM respectively.

Identification of individual components of a commercial wheat germ acid phosphatase preparation.

Wheat germ acid phosphatase (WGAP) is a commercial preparation of partially purified protein commonly used in laboratory settings for non-specific enzymatic dephosphorylation. It is known that these preparations contain multiple phosphatase isozymes and are still relatively crude. This study therefore aimed to identify the protein components of a commercial preparation of wheat germ acid phosphatase using mass spectroscopy and comparative genomics. After one post-purchase purification step, the most prevalent fifteen proteins in the mixture included heat shock proteins, beta-amylases, glucoseribitol dehydrogenases, enolases, and an aminopeptidase. While not among the most abundant components, eight unique dephosphorylation enzymes were also present including three purple acid phosphatases. Furthermore, it is shown that some of these correspond to previously isolated isozymes; one of which has been also previously shown by transcriptome data to be overexpressed in wheat seeds. In summary, this study identified the major components of WGAP including phosphatases and hypothesizes the most active components towards a better understanding of this commonly used laboratory tool.

Kinetic and thermodynamic studies of novel acid phosphates extracted from Cichorium intybus seedlings.

Herein for the first time a novel acid phosphatase from the seedlings of Cichorium intybus was purified to homogeneity by using various chromatographic techniques (salt precipitation, ion exchange, size exclusion and affinity chromatography) and thermodynamically characterized. The molecular mass of purified enzyme (66 kDa) was determined by SDS-PAGE under denaturing and non-denaturing conditions and by gel-filtration confirmed as dimer of molecular mass 130 kDa. The Michaelis-Menten (Km) constant for -p-NPP (0.3 mM) and (7.6 µmol/min/mg) Vmax. The enzyme was competitively inhibited by phosphate, molybdate and vanadate. Phenyl phosphate, ɑ and ß-glycero-phosphate and-p-NPP were found to be good substrate. When temperature increased from (55 °C to 75 °C), the deactivation rate constant (kd) was increased (0.1 to 4.6 min-1) and half- life was decreased from 630 min to 15 min. Various thermal denaturation parameters; change in enthalpy (ΔH°), change in entropy (ΔS°) and change in free energy (ΔG°) were found 121.93 KJ·mol-1, 72.45 KJ·mol-1 and 98.08 KJ·mol-1 respectively, confirming that acid phosphatase undergoes a significant process of unfolding during deactivation. The biochemical properties of acid phosphatase from C. intybus on the behalf of biological activity and its relationship to pH variations, thermal deactivation and kinetics parameters provide an insight into its novel features.

A novel acid phosphatase from cactus (Opuntia megacantha Salm-Dyck) cladodes: Purification and biochemical characterization of the enzyme.

Acid phosphatase (ACP) plays an important role in regulating phosphate nutrition in plants. Herein, for the first time, a novel ACP from Opuntia megacantha Salm-Dyck cladodes was purified to homogeneity and biochemically characterized. Specific activity of 8.78 U/mg was obtained with 11.29-fold purification and 15% yield. ACP was purified as monomer with molecular weight of 44 kDa as determined by SDS-PAGE under denaturing and nondenaturing conditions. Optimum pH and temperature for ACP activity was 5.5 and 60 °C, respectively. Thermodynamic parameters (Ea, ΔH, ΔG and ΔS) were also determined. ACP activity was stimulated by Ca2+, strongly inhibited by Cu2+ and Fe3+, and moderately inhibited by Mg2+ and Zn2+. Br-, CN-, F-, I- and N3- weakly inhibited ACP activity, where more than 70% of enzyme activity was remained at 5 mM. In addition, effect of ß-ME, Cys, DTT, EDTA, H2O2, PMSF, SDS and TX-100 on ACP activity was investigated. km, Vmax, kcat and kcat/km of ACP for p-NPP were found to be 0.09 mM, 2.75 U/mL, 9.60 s-1 and 106.67 s-1 mM-1, respectively. The biochemical properties of ACP from Opuntia megacantha Salm-Dyck cladodes provide novel features with other plant ACPs and basic knowledge of ACP in Opuntia species.

Engineering Two-Dimensional Pd Nanoplates with Exposed Highly Active {100} Facets Toward Colorimetric Acid Phosphatase Detection.

Enzyme-like activity and efficiency of nanomaterials are strongly controlled by their size, composition, and structure, and hence the structural parameters need to be optimized. Here, we report that two-dimensional Pd nanoplates enclosed by {100}-facets [{100}PdSP@rGO] exhibit substantially enhanced intrinsic oxidase-like activities relative to the {111}-facets ones and Pd nanocubes in catalyzing the chromogenic reaction of 3,3',5,5'-tetramethylbenzidine. By taking ascorbic acid 2-phosphate as the substrate, which transforms to ascorbic acid in the presence of acid phosphatase (ACP), the {100}PdSP@rGO could be used as an efficient nanozyme for colorimetric ACP detection without resorting to destructive H2O2. A good linear relationship from 0.01 to 6.0 mU/mL with a detection limit of 8.3 µU/mL is obtained, which is better than most previously reported ACP assays. Importantly, the excellent assay performance could be successfully applied to ACP determination in serum samples with high accuracy. This study demonstrates that the enzyme-like activity of nanomaterials could be finely tuned simultaneously by controlling their exposed crystal facets and high specific surface area.

The use of crime scene detection dogs to locate semen stains on different types of fabric.

In sexual assault cases, the detection and identification of semen is extremely important as this type of evidence can be used as a source for investigative leads and contributes to case evidence. However, the detection of semen stains is often difficult, even indoors, because of different (environmental) factors, such as substrate type, coloured items and large search areas. In 2015, a project was initiated by the Dutch police to evaluate the feasibility of the use of detection dogs to locate semen stains in forensic practise. Since promising results were obtained, here, a double-blind study was designed to investigate how these detection dogs can optimally be implemented in the current work flow of crime scene investigators and to compare the dog's sensitivity and specificity with current detection methods. The performance of the detection dogs was compared to three commonly used detection methods for semen, (i) forensic light sources (FLS), (ii) the RSID semen field kit and (iii) the enzymatic Acid Phosphatase (AP)-test on semen deposited at different types of fabrics. A 100% sensitivity and specificity for the detection of semen stains using the detection dogs was obtained, compared to an overall sensitivity and specificity of 76.3% and 100% for FLS, 81.6% and 100% for RSID-test, and 92.1% and 100% for AP-test, respectively. Especially, on fabrics with a pattern or interfering fluorescent properties, detection dogs demonstrated to be of additional value to locate the semen stains. We recommend to use the following order of testing, FLS, detection dog, AP-test and RSID test in a forensic workflow.

Introducing bifunctional metal-organic frameworks to the construction of a novel ratiometric fluorescence sensor for screening acid phosphatase activity.

Restricted to single sensing mechanisms, most luminescent metal-organic framework (LMOF)-based sensors were constructed for detection of limited targets. Here, a new biosensor is described for screening acid phosphatase (ACP) activity via bifunctional NH2-MIL-101 MOFs acting as both fluorescent indicator and biomimetic catalyst. NH2-MIL-101 possesses an inherent fluorescence emission at 456 nm (F456). As a peroxidase-like nanozyme, it catalyzes oxidation of o-phenylenediamine (OPD) by H2O2 to generate fluorescent 2,3-diaminophenazine with the maximum emission at 556 nm (F556). Upon introducing NH2-MIL-101 into a mixture of OPD and H2O2, F456 is quenched, while F556 increases. The ACP sensing is based on pyrophosphate ion (PPi) mediated fluorescence tuning of the NH2-MIL-101/OPD/H2O2 system. PPi inhibits the NH2-MIL-101 catalytic ability by specific binding to its Fe center, while ACP addition recovers the activity by hydrolyzing PPi. Upon addition of PPi and ACP into the NH2-MIL-101/OPD/H2O2 system, a ratiometric luminescence signal (F556/F456) is obtained, and a ratiometric fluorescent sensor can be developed for the sensitive detection of PPi and for screening ACP activity. Plots of F556/F456 vs. ACP concentration were linear over 0.01-30 U/L, with a detection limit of 0.005 U/L. The proposed sensor was successfully used for ACP detection in serum samples. This ratiometric fluorescence assay will open new applications for LMOF-based biosensors.

The acid phosphatase Pho5 of Saccharomyces cerevisiae is not involved in polyphosphate breakdown.

Inorganic polyphosphate is involved in architecture and functioning of yeast cell wall. The strain of Saccharomyces cerevisiae constitutively overexpressing acid phosphatase Pho5 was constructed for studying the Pho5 properties and its possible participation in polyphosphate metabolism. The parent strain was transformed by the vector carrying the PHO5 gene under a strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae. The culture liquid and biomass of transformant strain contained approximately equal total acid phosphatase activity. The levels of acid phosphatase activity associated with the cell wall and culture liquid increased in the transformant strain compared to the parent strain ~ 10- and 20-fold, respectively. The Pho5 preparation (specific activity of 46 U/mg protein and yield of 95 U/L) was obtained from culture liquid of overproducing strain. The overproducing strain had no changes in polyphosphate level. The activity of Pho5 with long-chained polyP was negligible. We concluded that Pho5 is not involved in polyphosphate metabolism. Purified Pho5 showed a similar activity with p-nitrophenylphosphate, ATP, ADP, glycerophosphate, and glucose-6-phosphate. The substrate specificity of Pho5 and its extracellular localization suggest its function: the hydrolysis of organic compounds with phosphoester bonds at phosphate limitation.

Lectin AtGAL1 interacts with high-mannose glycoform of the purple acid phosphatase AtPAP26 secreted by phosphate-starved Arabidopsis.

Among 29 predicted Arabidopsis purple acid phosphatases (PAPs), AtPAP26 functions as the principle extracellular and intracellular PAP isozyme that is upregulated to recycle and scavenge Pi during Pi-deprivation or leaf senescence. Our companion paper documented the copurification of a secreted, high-mannose AtPAP26-S2 glycoform with AtGAL1 (At1g78850), a Pi starvation-inducible (PSI), and Galanthus nivalis agglutinin-related (mannose-binding) and apple domain lectin. This study tests the hypothesis that AtGAL1 binds AtPAP26-S2 to modify its enzymatic properties. Far-western immunodot blotting established that AtGAL1 readily associates with AtPAP26-S2 but not the low mannose AtPAP26-S1 glycoform nor other secreted PSI PAPs (i.e., AtPAP12 or AtPAP25). Analytical gel filtration indicated that 55-kDa AtGAL1 and AtPAP26-S2 polypeptides associate to form a 112-kDa heterodimer. Microscopic imaging of transiently expressed, fluorescent protein-tagged AtGAL1, and associated bimolecular fluorescence complementation assays demonstrated that (a) like AtPAP26, AtGAL1 also localizes to lytic vacuoles of Pi-deprived Arabidopsis and (b) both proteins interact in vivo. AtGAL1 preincubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26-S2 but not AtPAP26-S1. We hypothesize that AtGAL1 plays an important role during Pi deprivation through its interaction with mannose-rich glycans of AtPAP26-S2 and consequent positive impact on AtPAP26-S2 activity and stability.

A glycoform of the secreted purple acid phosphatase AtPAP26 co-purifies with a mannose-binding lectin (AtGAL1) upregulated by phosphate-starved Arabidopsis.

The purple acid phosphatase AtPAP26 plays a central role in Pi-scavenging by Pi-starved (-Pi) Arabidopsis. Mass spectrometry (MS) of AtPAP26-S1 and AtPAP26-S2 glycoforms secreted by -Pi suspension cells demonstrated that N-glycans at Asn365 and Asn422 were modified in AtPAP26-S2 to form high-mannose glycans. A 55-kDa protein that co-purified with AtPAP26-S2 was identified as a Galanthus nivalis agglutinin-related and apple domain lectin-1 (AtGAL1; At1g78850). MS revealed that AtGAL1 was bisphosphorylated at Tyr38 and Thr39 and glycosylated at four conserved Asn residues. When AtGAL was incubated in the presence of a thiol-reducing reagent prior to immunoblotting, its cross-reactivity with anti-AtGAL1-IgG was markedly attenuated (consistent with three predicted disulfide bonds in AtGAL1's apple domain). Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating posttranscriptional control of AtGAL1 expression. Growth of a -Pi atgal1 mutant was unaffected, possibly due to compensation by AtGAL1's closest paralog, AtGAL2 (At1g78860). Nevertheless, AtGAL1's induction by numerous stresses combined with the broad distribution of AtGAL1-like lectins in diverse species implies an important function for AtGAL1 orthologs within the plant kingdom. We hypothesize that binding of AtPAP26-S2's high-mannose glycans by AtGAL1 enhances AtPAP26 function to facilitate Pi-scavenging by -Pi Arabidopsis.

Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline.

Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds 'Generally Recognised As Safe' status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate; (2) growth as short fragments, as for Escherichia coli; (3) high efficiency of transformation by electroporation; and (4) secretion of proteins into the culture medium. In this study, we specifically focused our efforts on an exploration of the use of the Sec secretory pathway to export heterologous proteins in a S. rimosus host. We aimed to develop a genetic tool kit for S. rimosus and to evaluate the extracellular production of target heterologous proteins of this industrial host. This study demonstrates that S. rimosus can produce the industrially important enzyme phytase AppA extracellularly, and analogous to E. coli as a host, application of His-Tag/Ni-affinity chromatography provides a simple and rapid approach to purify active phytase AppA in S. rimosus. We thus demonstrate that S. rimosus can be used as a potential alternative protein expression system.

Expression, purification, and characterization of a novel acid phosphatase that displays protein tyrosine phosphatases activity from Metarhizium anisopliae strain CQMa102.

The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system. Our group has purified a type of acid phosphatase that could specifically dephosphorylate trans-Golgi p230 in vitro. In order to study this phosphatase further, we have identified and cloned the phosphatase gene from a locust specific Metarhizium anisopliae Strain CQMa102. The CQMa102 phosphatase was expressed in Pichia pastoris to verify its protease activity. The molecular weight (MW) and the isoelectric point (pI) of the phosphatase were about 85 kDa and 6.15, respectively. Substrate specificity evaluation showed that the purified enzyme exhibited high activity on O-phospho-L-tyrosine. At its optimal pH of 6.5 and optimum temperature of 70 °C, the protein showed the highest activity respectively. It can be activated by Ca2+, Mg2+, Mn2+, Ba2+, Co2+ and phosphate analogs, but inhibited by Zn2+, Cu2+, fluoride, dithiothreitol, ß-mercaptoethanol and N-ethylmaleimide.

Alumina nanoparticle-assisted enzyme refolding: A versatile methodology for proteins renaturation.

We present a high-yield method for the renaturation of negatively charged enzymes. The approach is based on the use of alumina nanoparticles, which after electrostatic interaction with denatured protein molecules, prevent their aggregation and make the process of refolding controllable. The method, demonstrated by the renaturation of several enzymes, is efficient, rapid, employs a minimal amount of reagents and even can be applied to renature mixture of the denatured enzymes.

Isolation of Autolysosomes from Tobacco BY-2 Cells.

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H+-ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli.

The phytase gene appAS was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppAS) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppAS contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appAS was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppAP and rAppAE, respectively. Purified glycosylated rAppAP and nonglycosylated rAppAE had specific activity of 967 and 2982 U mg(-1), respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppAE, rAppAP was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppAP was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppAE. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.

Purification and biochemical characterisation of acid phosphatase-I from seeds of Nelumbo nucifera.

Acid phosphatase-I (Apase-I) from seeds of Nelumbo nucifera was purified to electrophoretic homogeneity by combination of ammonium sulfate precipitation, size-exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kDa under reducing and non-reducing conditions, indicating that the enzyme was a monomer. The purified enzyme showed maximum activity at 50°C and at pH 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 µM, 10 µmol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn(2+), W(2+); weakly inhibited by Cu(2+), Mo(2+) and Cr(6+) and moderately activated by Mg(2+). The enzyme was shown to be thermolabile as it lost 50% of its activity at 50°C after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase.

Hydrodynamic voltammetry as a rapid and simple method for evaluating soil enzyme activities.

Soil enzymes play essential roles in catalyzing reactions necessary for nutrient cycling in the biosphere. They are also sensitive indicators of ecosystem stress, therefore their evaluation is very important in assessing soil health and quality. The standard soil enzyme assay method based on spectroscopic detection is a complicated operation that requires the removal of soil particles. The purpose of this study was to develop a new soil enzyme assay based on hydrodynamic electrochemical detection using a rotating disk electrode in a microliter droplet. The activities of enzymes were determined by measuring the electrochemical oxidation of p-aminophenol (PAP), following the enzymatic conversion of substrate-conjugated PAP. The calibration curves of ß-galactosidase (ß-gal), ß-glucosidase (ß-glu) and acid phosphatase (AcP) showed good linear correlation after being spiked in soils using chronoamperometry. We also performed electrochemical detection using real soils. Hydrodynamic chronoamperometry can be used to assess the AcP in soils, with a detection time of only 90 s. Linear sweep voltammetry was used to measure the amount of PAP released from ß-gal and ß-glu by enzymatic reaction after 60 min. For the assessment of soil enzymes, the results of hydrodynamic voltammetry assay compared favorably to those using a standard assay procedure, but this new procedure is more user-friendly, rapid and simple.

Identification of a class B acid phosphatase in Haemophilus parasuis

An acid phosphatase activity was detected in the supernatant of Haemophilus parasuis, a Gram-negative pleomorphic bacillus and the causative agent of Glässer’s disease in pigs. To identify the gene responsible for the secreted activity, a genomic library of H. parasuis strain ER-6P was produced in Escherichia coli. Screening of the library allowed identification of two homologs to known phosphatases: PgpB and AphA. PgpB was predicted to be located in the bacterial membrane through six transmembrane domains while AphA was predicted to have a signal peptide. The aphA gene was cloned and expressed in E. coli. Characterization of H. parasuis AphA indicated that this protein belongs to the class B nonspecific acid phosphatases. AphA contained sequence signatures characteristic of this family of phosphatases and its activity was inhibited by EDTA. The optimal pH of recombinant AphA differed from that of the phosphatase activity found in H. parasuis supernatants. In addition, the phosphatase activity from H. parasuis supernatants was not inhibited by EDTA, indicating that H. parasuis AphA does not account for the phosphatase activity observed in the supernatants. Our results demonstrate the presence of a class B acid phosphatase (AphA) in H. parasuis and suggest that the bacterium would also secrete another, as yet unidentified phosphatase (AU)

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Immobilization of acid phosphatase from Vigna aconitifolia seeds on chitosan beads and its characterization.

Acid phosphatase isolated from Vigna aconitifolia seeds was immobilized onto glutaraldehyde activated chitosan beads by crosslinking method. Chitosan beads activated with 2% of glutaraldehyde have demonstrated maximum immobilization yield (∼ 83%). The immobilized enzyme showed optimum activity at pH 7.0, while soluble form was maximally active in acidic range (pH 5.0). With respect to free form, immobilized acid phosphatase showed better activity in alkaline range. On the other side, immobilization does not affect the optimum temperature range i.e., both, soluble and immobilized acid phosphatase exhibited maximum activity at 60 °C. The Km and Vmax values for the immobilized enzyme were calculated to be 0.37 mM and 13.5 U/mg. The immobilization on chitosan beads enhanced the shelf life of acid phosphatase. The immobilized enzyme retained its more than 50% hydrolytic activity for approximately two months. The immobilized acid phosphatase was reusable for more than 40 cycles of reaction.

Isolation and enzymatic properties of a nonspecific acid phosphatase from Vigna aconitifolia seeds.

Acid phosphatase (EC 3.1.3.2) from Vigna aconitifolia seeds was purified to apparent homogeneity by using ammonium sulfate fractionation and cation-exchange chromatography [carboxymethyl (CM) cellulose]. The enzyme was 228-fold purified with 14.6% recovery. Analytical gel filtration chromatography on Sephadex G-200 column showed that Mr of native enzyme was 58 kDa and denaturing PAGE demonstrated that it was made up of two subunits of 24 and 27 kDa. The enzyme showed its optimum activity at pH 5.0 and 60°C. It exhibited broad substrate specificity and showed a higher specificity constant for para-nitrophenyl phosphate, Na ß-naphthyl phosphate, and adenosine monophosphate (AMP). Cu²âº, Mo6⁺, Fe³âº, phosphate, and fluoride ions were reported as strong inhibitors for the enzyme. Active site study for the enzyme demonstrated that tryptophan and aspartic acid may be important for the catalysis.