Evaluation of the Effectiveness of Innovative Sorbents in Restoring Enzymatic Activity of Soil Contaminated with Bisphenol A (BPA).

As part of the multifaceted strategies developed to shape the common environmental policy, considerable attention is now being paid to assessing the degree of environmental degradation in soil under xenobiotic pressure. Bisphenol A (BPA) has only been marginally investigated in this ecosystem context. Therefore, research was carried out to determine the biochemical properties of soils contaminated with BPA at two levels of contamination: 500 mg and 1000 mg BPA kg-1 d.m. of soil. Reliable biochemical indicators of soil changes, whose activity was determined in the pot experiment conducted, were used: dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, arylsulfatase, and ß-glucosidase. Using the definition of soil health as the ability to promote plant growth, the influence of BPA on the growth and development of Zea mays, a plant used for energy production, was also tested. As well as the biomass of aerial parts and roots, the leaf greenness index (SPAD) of Zea mays was also assessed. A key aspect of the research was to identify those of the six remediating substances-molecular sieve, zeolite, sepiolite, starch, grass compost, and fermented bark-whose use could become common practice in both environmental protection and agriculture. Exposure to BPA revealed the highest sensitivity of dehydrogenases, urease, and acid phosphatase and the lowest sensitivity of alkaline phosphatase and catalase to this phenolic compound. The enzyme response generated a reduction in the biochemical fertility index (BA21) of 64% (500 mg BPA) and 70% (1000 mg BPA kg-1 d.m. of soil). The toxicity of BPA led to a drastic reduction in root biomass and consequently in the aerial parts of Zea mays. Compost and molecular sieve proved to be the most effective in mitigating the negative effect of the xenobiotic on the parameters discussed. The results obtained are the first research step in the search for further substances with bioremediation potential against both soil and plants under BPA pressure.

Transcription level and phylogeny analyses of Chlamydomonas reinhardtii arylsulfatases.

Sulfur is a required macroelement for all organisms, and sulfate deficiency causes growth and developmental defects. Arylsulfatases (ARS) hydrolyze sulfate from sulfate esters and make sulfate bioavailable for plant uptake. These enzymes are found in microorganisms and animals; however, plant genomes do not encode any ARS gene. Our database searches found nineteen ARS genes in the genome of Chlamydomonas reinhardtii. Among these, ARS1 and ARS2 were studied in the literature; however, the remaining seventeen gene models were not studied. Our results show that putative polypeptide sequences of the ARS gene models all have the sulfatase domain and sulfatase motifs found in known ARSs. Phylogenetic analyses show that C. reinhardtii proteins are in close branches with Volvox carterii proteins while they were clustered in a separate group from Homo sapiens and bacterial species (Pseudomonas aeruginosa and Rhodopirellula baltica SH1), except human Sulf1, Sulf2, and GNS are clustered with algal ARSs. RT-PCR analyses showed that transcription of ARS6, ARS7, ARS11, ARS12, ARS13, ARS17, and ARS19 increased under sulfate deficiency. However, this increase was not as high as the increase seen in ARS2. Since plant genomes do not encode any ARS gene, our results highlight the importance of microbial ARS genes.

Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni.

BACKGROUND: Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. RESULTS: The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). CONCLUSIONS: This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).

The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wild-type and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate.

New clinical and molecular evidence linking mutations in ARSG to Usher syndrome type IV.

In murine and canine animal models, mutations in the Arylsulfatase G gene (ARSG) cause a particular lysosomal storage disorder characterized by neurological phenotypes. Recently, two variants in the same gene were found to be associated with an atypical form of Usher syndrome in humans, leading to visual and auditory impairment without the involvement of the central nervous system. In this study, we identified three novel pathogenic variants in ARSG, which segregated recessively with the disease in two families from Portugal. The probands were affected with retinitis pigmentosa and sensorineural hearing loss, generally with an onset of symptoms in their fourth decade of life. Functional experiments showed that these pathogenic variants abolish the sulfatase activity of the Arylsulfatase G enzyme and impede the appropriate lysosomal localization of the protein product, which appears to be retained in the endoplasmic reticulum. Our data enable to definitely confirm that different biallelic variants in ARSG cause a specific deaf-blindness syndrome, by abolishing the activity of the enzyme it encodes.

Arylsulfatase K inactivation causes mucopolysaccharidosis due to deficient glucuronate desulfation of heparan and chondroitin sulfate.

Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of HS and CS metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of mucopolysacharidose (MPS), which we designate MPS type IIB.

Implementation of an Affordable Method for MPS Diagnosis from Urine Screening to Enzymatic Confirmation: Results of a Pilot Study in Morocco.

BACKGROUND: Rapid and accurate diagnosis of mucopolysaccharidoses (MPS) is still a challenge due to poor access to screening and diagnostic methods and to their extensive clinical heterogeneity. The aim of this work is to perform laboratory biochemical testing for confirming the diagnosis of mucopolysaccharidosis (MPS) for the first time in Morocco. METHODS: Over a period of twelve months, 88 patients suspected of having Mucopolysaccharidosis (MPS) were referred to our laboratory. Quantitative and qualitative urine glycosaminoglycan (GAG) analyses were performed, and enzyme activity was assayed on dried blood spots (DBS) using fluorogenic substrates. Enzyme activity was measured as normal, low, or undetectable. RESULTS: Of the 88 patients studied, 26 were confirmed to have MPS; 19 MPS I (Hurler syndrome; OMIM #607014/Hurler-Scheie syndrome; OMIM #607015), 2 MPS II (Hunter syndrome; OMIM #309900), 2 MPS IIIA (Sanfilippo syndrome; OMIM #252900), 1 MPS IIIB (Sanfilippo syndrome; OMIM #252920) and 2 MPS VI (Maroteaux-Lamy syndrome; OMIM #253200). Parental consanguinity was present in 80.76% of cases. Qualitative urinary glycosaminoglycan (uGAGs) assays showed abnormal profiles in 31 cases, and further quantitative urinary GAG evaluation and Thin Layer Chromatography (TLC) provided important additional information about the likely MPS diagnosis. The final diagnosis was confirmed by specific enzyme activity analysis in the DBS samples. CONCLUSIONS: The present study shows that the adoption of combined urinary substrate analysis and enzyme assays using dried blood spots can facilitate such diagnosis, offer an important tool for an appropriate supporting care, and a specific therapy, when available.

Alteration of enzyme activities and functional diversity of a soil contaminated with copper and arsenic.

Copper (Cu) mining has to address a critical environmental issue related to the disposal of heavy metals and metalloids (HMs). Due to their deleterious effects on living organisms, Cu and arsenic (As) have gained global attention, and thus their monitoring in the environment is an important task. The aims of this study were: 1) to evaluate the alteration of soil enzyme activities (EAs) and soil microbial functional diversity with Cu/As contamination, and 2) to select the most reliable biochemical indicators of Cu/As contamination. A twelve-week soil experiment was performed with four increasing levels of Cu, As, and Cu/As from 150/15 to 1000/100 mg Cu/As kg-1. Soil enzyme activities and soil community-level physiological profile (CLPP) using MicroResp™ were measured during the experiment. Results showed reduced EAs over time with increasing Cu and Cu/As levels. The most Cu-sensitive EAs were dehydrogenase, acid phosphatase, and arylsulfatase, while arginine ammonification might be related to the resilience of soil microbial communities due to its increased activity in the last experimental times. There was no consistent response to As contamination with reduced individual EAs at specific sampling times, being urease the only EA negatively affected by As. MicroResp™ showed reduced carbon (C) substrate utilization with increasing Cu levels indicating a community shift in C acquisition. These results support the use of specific EAs to assess the environmental impact of specific HMs, being also the first assessment of EAs and the use of CLPP (MicroResp™) to study the environmental impact in Cu/As contaminated soils.

Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (ßleaving groupobs-Enz = -0.33) than the uncatalyzed reaction (ßleaving groupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δßleaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

Comprehensive kinetic and substrate specificity analysis of an arylsulfatase from Helix pomatia using mass spectrometry.

Sulfatases hydrolyze sulfated metabolites to their corresponding alcohols and are present in all domains of life. These enzymes have found major application in metabolic investigation of drugs, doping control analysis and recently in metabolomics. Interest in sulfatases has increased due to a link between metabolic processes involving sulfated metabolites and pathophysiological conditions in humans. Herein, we present the first comprehensive substrate specificity and kinetic analysis of the most commonly used arylsulfatase extracted from the snail Helix pomatia. In the past, this enzyme has been used in the form of a crude mixture of enzymes, however, recently we have purified this sulfatase for a new application in metabolomics-driven discovery of sulfated metabolites. To evaluate the substrate specificity of this promiscuous sulfatase, we have synthesized a series of new sulfated metabolites of diverse structure and employed a mass spectrometric assay for kinetic substrate hydrolysis evaluation. Our analysis of the purified enzyme revealed that the sulfatase has a strong preference for metabolites with a bi- or tricyclic aromatic scaffold and to a lesser extent for monocyclic aromatic phenols. This metabolite library and mass spectrometric method can be applied for the characterization of other sulfatases from humans and gut microbiota to investigate their involvement in disease development.

Interactive effect of salinity and cadmium toxicity on soil microbial properties and enzyme activities.

Salinity has been proposed to increase the mobility and availability of heavy metals, with a potentially significant consequence for greater metal toxicity. However, the interactive effect of salinity and metal pollution on soil microbial properties and functions is still unknown. This investigation was performed to examine the response of several soil microbial properties and processes to the combined salinity and cadmium (Cd) toxicity in a clay loam soil amended with plant residue. The NaCl salt (0, 32.5 and 78.3 mM NaCl kg-1 soil), Cd (0 and 30 mg kg-1 soil) and alfalfa residue (0 and 1%) were added to the soil and the mixtures were incubated for 90 days under standard laboratory conditions (25 ±â€¯1 °C and 70% of water holding capacity). Similar treatments without residue addition were also included in the experimental arrangement. Salinity increased soil Cd availability and toxicity, and subsequently decreased soil microbial respiration rate, microbial biomass and enzyme activity. The negative effect of increasing salinity on soil microbial and biochemical properties was stronger in Cd-polluted than unpolluted soils and at high than low salinity levels. The declines in soil microbial attributes and enzyme activity were linearly related to the concentration of soil available Cd. Nevertheless, the negative effect of salinity was reduced with addition of alfalfa residue in Cd-polluted soils. The interactive effect of Cd and NaCl was synergistic in residue-unamended soils, but antagonistic in residue-amended soils. It is concluded that (i) the multiple stresses induced by salinity and Cd pollution may synergistically affect soil microbial processes and attributes and (ii) application of organic residues has a high potential for lowering the synergistic effect of salinity in Cd-polluted environments and improving the important microbial indicators of soil quality.

A homozygous founder missense variant in arylsulfatase G abolishes its enzymatic activity causing atypical Usher syndrome in humans.

PURPOSE: We aimed to identify the cause of disease in patients suffering from a distinctive, atypical form of Usher syndrome. METHODS: Whole-exome and genome sequencing were performed in five patients from three families of Yemenite Jewish origin, suffering from distinctive retinal degeneration phenotype and sensorineural hearing loss. Functional analysis of the wild-type and mutant proteins was performed in human fibrosarcoma cells. RESULTS: We identified a homozygous founder missense variant, c.133G>T (p.D45Y) in arylsulfatase G (ARSG). All patients shared a distinctive retinal phenotype with ring-shaped atrophy along the arcades engirdling the fovea, resulting in ring scotoma. In addition, patients developed moderate to severe sensorineural hearing loss. Both vision and hearing loss appeared around the age of 40 years. The identified variant affected a fully conserved amino acid that is part of the catalytic site of the enzyme. Functional analysis of the wild-type and mutant proteins showed no basal activity of p.D45Y. CONCLUSION: Homozygosity for ARSG-p.D45Y in humans leads to protein dysfunction, causing an atypical combination of late-onset Usher syndrome. Although there is no evidence for generalized clinical manifestations of lysosomal storage diseases in this set of patients, we cannot rule out the possibility that mild and late-onset symptoms may appear.

A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity.

Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as ß-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of ß-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC 3.1.6.1). Due to the action of arylsulfatase, an unpleasant "cowshed-like" off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.

Biochemical and microbiological activity of soil contaminated with o-cresol and biostimulated with Perna canaliculus mussel meal.

The choice of the study subject was a consequence of the growing interest in volatile organic compounds which are strongly dispersed in the environment. The knowledge of o-cresol's capability for being broken down by bacteria should be supplemented by studies aimed at determining the biochemical and microbiological activity of soils. o-Cresol was applied at the following rates: 0, 0.1, 1, 10, and 50 mg of o-cresol kg-1 d.m. of soil to determine its effect on the biological properties of soil. The activity of dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, arylsulfatase, and ß-glucosidase, the eight groups of microorganism counts, was determined in soil samples after 45 days and the barley yield was determined. Preventive biostimulation with Perna canaliculus mussel meal, illustrated by means of the index of fertility (IF), was conducted in order to eliminate the adverse effect of o-cresol. The soil and crop resistance index (RS) was used to illustrate the response of barley, and R:S-the rhizosphere effect index was used to determine the effect of the crop on the enzymatic activity of soil. o-Cresol had a beneficial effect on the biological activity of soil at an acceptable rate of 0.1 and 1 mg kg-1 d.m. of soil, and it became its inhibitor after being applied at 10 and 50 mg kg-1 d.m. of soil, which also brought about a decrease in the resistance of spring barley. Dehydrogenases are the most sensitive, and catalase is the least sensitive, to the pressure of o-cresol in soil. Mussel meal can be recommended as a biostimulator of soil fertility. It also eliminated the negative effect of o-cresol on its biological activity.

Synergistic interaction of glyceraldehydes-3-phosphate dehydrogenase and ArsJ, a novel organoarsenical efflux permease, confers arsenate resistance.

Microbial biotransformations are major contributors to the arsenic biogeocycle. In parallel with transformations of inorganic arsenic, organoarsenicals pathways have recently been recognized as important components of global cycling of arsenic. The well-characterized pathway of resistance to arsenate is reduction coupled to arsenite efflux. Here, we describe a new pathway of arsenate resistance involving biosynthesis and extrusion of an unusual pentavalent organoarsenical. A number of arsenic resistance (ars) operons have two genes of unknown function that are linked in these operons. One, gapdh, encodes the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The other, arsJ, encodes a major facilitator superfamily (MFS) protein. The two genes were cloned from the chromosome of Pseudomonas aeruginosa. When expressed together, but not alone, in Escherichia coli, gapdh and arsJ specifically conferred resistance to arsenate and decreased accumulation of As(V). Everted membrane vesicles from cells expressing arsJ accumulated As(V) in the presence of purified GAPDH, D-glceraldehylde 3-phosphate (G3P) and NAD(+) . GAPDH forms the unstable organoarsenical 1-arseno-3-phosphoglycerate (1As3PGA). We propose that ArsJ is an efflux permease that extrudes 1As3PGA from cells, where it rapidly dissociates into As(V) and 3-phosphoglycerate (3PGA), creating a novel pathway of arsenate resistance.

Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation.

Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic L-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions.

High-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins.

High-throughput estimation of specific activities of an enzyme and its mutants in a group (enzyme/mutants) in cell lysates via high-throughput assay of their activities and separate immunoturbidimetric assay (ITA) of their proteins was proposed. Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) served as two models. ITA employed 0.75 mg of antisera against PAAS or BFU as the reference in 96-well microplates to measure the difference of extinction at 340 and 700 nm. According to the calibration curve, ITA quantified the reference from 0.40 to about 2.4 µg. The consistency among the abundance of enzyme/mutants through ITA of proteins in cell lysates prepared under the same conditions supported their consistent immunological reactivity to the antisera. Specific activities of PAAS/mutants or BFU/mutants in cell lysates through ITA of proteins showed excellent proportionality to those carefully determined after purification. Receiver-operating-characteristic (ROC) analysis of specific activities through ITA of proteins gave a higher area-under-curve than those for ROC analyses of other activity indices, which allowed the recognition of a PAAS/mutant of 50% higher activity after cell amplification in high-throughput mode. Therefore, ITA of enzyme/mutants as proteins is promising to estimate their specific activities in cell lysates in high-throughput mode for quantitative comparison.

Detection, production, and application of microbial arylsulfatases.

Arylsulfatases are enzymes which catalyze the hydrolysis of arylsulfate ester bonds to release a free sulfonate. They are widespread in nature and are found in microorganisms, most animal and human tissues, and plant seeds. However, this review focuses on arylsulfatases from microbial origin and gives an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the production of microbial arylsulfatases using wild-type organisms as well as the recombinant production using Escherichia coli and Kluyveromyces lactis as expression hosts is discussed. Finally, various potential applications of these enzymes are reviewed.

Homologous expression and biochemical characterization of the arylsulfatase from Kluyveromyces lactis and its relevance in milk processing.

The industrial manufacturing process of lactose-free milk products depends on the application of commercial ß-galactosidase (lactase) preparations. These preparations are often obtained from Kluyveromyces lactis. There is a gene present in the genome of K. lactis which should encode for an enzyme called arylsulfatase (EC 3.1.6.1). Therefore, this enzyme could also be present in ß-galactosidase preparations. The arylsulfatase is suspected of being responsible for an unpleasant "cowshed-like" off-flavor resulting from the release of p-cresol from milk endogenous alkylphenol sulfuric esters. So far, no gene/functionality relationship is described. In addition, no study is available which has shown that arylsulfatase from K. lactis is truly responsible for the flavor generation. In this study, we cloned the putative arylsulfatase gene from K. lactis GG799 into the commercially available vector pKLAC2. The cloning strategy chosen resulted in a homologous, secretory expression of the arylsulfatase. We showed that the heretofore putative arylsulfatase has the desired activity with the synthetic substrate p-nitrophenyl sulfate and with the natural substrate p-cresol sulfate. The enzyme was biochemically characterized and showed an optimum temperature of 45-50 °C and an optimum pH of 9-10. Additionally, the arylsulfatase was activated by Ca(2+) ions and was inactivated by Zn(2+) ions. Moreover, the arylsulfatase was inhibited by p-cresol and sulfate ions. Finally, the enzyme was added to ultra-heat treated (UHT) milk and a sensory triangle test verified that the arylsulfatase from K. lactis can cause an unpleasant "cowshed-like" off-flavor.

Molecular characterization of arylsulfatase G: expression, processing, glycosylation, transport, and activity.

Arylsulfatase G (ARSG) is a recently identified lysosomal sulfatase that was shown to be responsible for the degradation of 3-O-sulfated N-sulfoglucosamine residues of heparan sulfate glycosaminoglycans. Deficiency of ARSG leads to a new type of mucopolysaccharidosis, as described in a mouse model. Here, we provide a detailed molecular characterization of the endogenous murine enzyme. ARSG is expressed and proteolytically processed in a tissue-specific manner. The 63-kDa single-chain precursor protein localizes to pre-lysosomal compartments and tightly associates with organelle membranes, most likely the endoplasmic reticulum. In contrast, proteolytically processed ARSG fragments of 34-, 18-, and 10-kDa were found in lysosomal fractions and lost their membrane association. The processing sites and a disulfide bridge between the 18- and 10-kDa chains could be roughly mapped. Proteases participating in the processing were identified as cathepsins B and L. Proteolytic processing is dispensable for hydrolytic sulfatase activity in vitro. Lysosomal transport of ARSG in the liver is independent of mannose 6-phosphate, sortilin, and Limp2. However, mutation of glycosylation site N-497 abrogates transport of ARSG to lysosomes in human fibrosarcoma cells, due to impaired mannose 6-phosphate modification.