Comparative study of esterases in deltamethrin and diazinon resistant Rhipicephalus microplus and Hyalomma anatolicum ticks collected from the Trans-Gangetic plains of India.

A comparative analysis of esterases in susceptible and resistant ticks revealed six types of esterases (EST-1b, EST-2b, EST-3b, EST-4b, EST-5b and EST-6b) in Rhipicephalus microplus and four types (EST-1h, EST-2h, EST-3h, EST-4h) in Hyalomma anatolicum using α-naphthyl acetate substrate. Inhibition studies with eserine sulfate, p-chloromercuribenzoate, copper sulphate and phenylmethylsulfonyl fluoride revealed a marked variation in band intensity between susceptible and resistant ticks, with the latter being more intense. Qualitative expression of EST-4b along with an extra band of EST-5b and EST-6b were indicative of deltamethrin and diazinon resistance in R. microplus, whereas qualitative expression of EST-4h was probably responsible for diazinon resistance in H. anatolicum. The data suggest that increased esterase activity may represent a detoxification strategy leading to the development of resistance in these tick populations.

Acaricide resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum collected from Haryana and Rajasthan states of India.

Acaricide resistance status of Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum ticks against deltamethrin and diazinon was assessed in Hisar (Haryana) and its adjoining district Churu (Rajasthan) using adult immersion test (AIT) and larval packet test (LPT). The mortality slope, LC50, LC95, 95 % confidence limit and resistance factor of field ticks were determined. Results showed that R. (B.) microplus ticks collected from Tohana (Hisar) were found resistant to both the acaricides while the ticks of Agroha (Hisar) were found to be susceptible using AIT. Similar results were observed by using standard method of LPT. Again, H. anatolicum tick isolates of Tara Nagar (Churu) were found susceptible whereas Churu tick isolates were found to be resistant using AIT for both the acaricides. LPT indicated susceptible status of H. anatolicum ticks collected from Churu as well as Tara Nagar for both the acaricides. The study warrants the need for strategic use of available acaricides to overcome the development of acaricide resistance in ticks.

Anticancer activity of two ruthenium(II)-DMSO-chalcone complexes: Comparison of cytotoxic, pro-apoptotic and antimetastatic potential.

PURPOSE: Recently, we reported the synthesis and characterization of two complexes of general formula cis-[Ru(S-DMSO)3(R-CO-CH=CH-R')Cl] (R = 2-hydroxyphenyl for both, R' = thiophene (1), 3-methyl thiophene (2)) that showed remarkable topoisomerase II inhibition and strong binding with DNA. The aim of this study was the investigation of cytotoxic properties of these complexes against a panel of human tumor cell lines, with elucidation of their anticancer mechanisms in HeLa cells. METHODS: Characterization of anticancer activity of the investigated ruthenium complexes 1 and 2 included analysis of cytotoxicity by MTT assay. Cell cycle phase disruption of HeLa cells treated with complexes 1 and 2 was analyzed by flow cytometry after propidium iodide (PI) staining. Annexin V-FITC/PI double staining and further flow cytometry analysis and acridine orange (AO)/ethidium bromide (EB) double staining and fluorescent microscopy were used to determine the apoptotic potential of the investigated ruthenium complexes. The inhibitory effect on gelatinases (MMP-2 and MMP-9) as an indication of possible antimetastatic potential was also analyzed using gelatine zymography. RESULTS: The 50% cell growth inhibition (IC50) values of the investigated complexes ranged between 22.9 and 76.8 µM, with complex 2 being more cytotoxic. Both complexes induced G2 phase cell cycle arrest and apoptosis in HeLa cells. Inhibitory effect of complex 2 on MMP-2 activity was detected. CONCLUSIONS: This work revealed the potential of the investigated Ru(II)-DMSO-chalcone complexes as anticancer agents with cytotoxic and pro-apoptotic activity and indicated complex 2 as leading compound for further chemical modifications and anticancer research.

Synthetic and natural antibacterial carbon adsorbents for clinical nuclear waste management.

Patients undergoing high-dose radioiodine ablation (RAI) therapy in Nuclear Medicine Department need to be isolated in a special designed ward for a few days. Large amount of clinical radioactive wastewater from patient body is produced during high-activity RAI therapy. The radioactive wastewater needs to store in a delay tank until the radioactivity decayed below acceptable limit before being discharged and indirectly limit the patient admission and treatment. This study is to propose an alternative antibacterial adsorbent for I-131 extraction from clinical radioactive wastewater at the nuclear medicine department using graphene oxide silver (GOAg) and bamboo activated carbon (BAC). The synthesised adsorbents and their sediments (filtered sample) were analysed using field emission scanning electron microscopy (FESEM) for morphological analysis and analysed using X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) and X-ray diffraction (XRD). XPS spectra for C 1s adsorbents show intensity peaks at 284.45 eV (C=C) and 285.3 eV (C-C) for GOAg and its sediments, and 284.35 eV (C-C), 287.00 eV (C=O), and 290.07 eV (π-π∗ transitions) for BAC and its sediments. FTIR spectra reveal various functional groups of adsorbents: C=C (1637.50772 cm-1), C=O (1340.48041 cm-1), and C-O-C (1031.88060 cm-1) for GOAg and its sediments, and C=C (1635.57897 cm-1), C-C (1257.54421 cm-1), and C-O (1188.10925 cm-1) for BAC and its sediments. XRD patterns exhibit peaks at 2θ = 27.82°, 29.39°, 32.24°, and 46.22°, which can be attributed to the (002) diffraction plane, (220) crystallographic plane, (111) plane of Ag2O, and (200) crystallographic plane, respectively, for GOAg and its sediments. Meanwhile, the peaks at 2θ = 26.56° and 42.41°, which correspond to (002) and (100) planes, respectively, for BAC and its sediments. The d-spacing and the crystallinity index of each adsorbent were also determined. The estimation of the remaining ß- particles during the adsorption of I-131 was carried out using PHITS. The finding of this study is beneficial for alternative radionuclide extractions technique from clinical radioactive wastewater in nuclear medicine.

Functional characterization of the ferroxidase, permease high-affinity iron transport complex from Candida albicans.

Saccharomyces cerevisiae expresses two proteins that together support high-affinity Fe-uptake. These are a multicopper oxidase, Fet3p, with specificity towards Fe²âº and a ferric iron permease, Ftr1p, which supports Fe-accumulation. Homologues of the genes encoding these two proteins are found in all fungal genomes including those for the pathogens, Candida albicans and Cryptococcus neoformans. At least one of these loci represents a virulence factor for each pathogen suggesting that this complex would be an appropriate pharmacologic target. However, the mechanism by which this protein pair supports Fe-uptake in any fungal pathogen has not been elucidated. Taking advantage of the robust molecular genetics available in S. cerevisiae, we identify the two of five candidate ferroxidases likely involved in high-affinity Fe-uptake in C. albicans, Fet31 and Fet34. Both localize to the yeast plasma membrane and both support Fe-uptake along with an Ftr1 protein, either from C. albicans or from S. cerevisiae. We express and characterize Fet34, demonstrating that it is functionally homologous to ScFet3p. Using S. cerevisiae as host for the functional expression of the C. albicans Fe-uptake proteins, we demonstrate that they support a mechanism of Fe-trafficking that involves channelling of the CaFet34-generated Fe³âº directly to CaFtr1 for transport into the cytoplasm.

Synthesis and characterization of Ru(II)-DMSO-Cl-chalcone complexes: DNA binding, nuclease, and topoisomerase II inhibitory activity.

The complexes of type cis-[Ru(S-DMSO)(3)(R-CO-CH═CH-R')Cl] (R = 2-hydroxyphenyl for all, R' = phenyl 1, naphthyl 2, anthracenyl 3, thiophene 4, 3-methyl thiophene 5) are synthesized and characterized using spectroscopic (IR, (1)H and (13)C NMR, and UV-vis) and single crystal X-ray diffraction techniques. Their crystal structures show the formation of both intermolecular and intramolecular H-bonding. The molecular assembly of complex 5 using secondary interactions provides a butterfly structure. The binding of complexes with calf thymus DNA is monitored using UV-vis spectral titrations. The binding interaction of complexes 1, 2, and 3 with DNA increases with increasing conjugation of aromatic rings. However, complexes 4 and 5 interact with DNA strongly. The emission from ethidium bromide (EB) bound DNA recorded in phosphate buffer solution (pH = 7.2) decreases by incremental addition of solution of the complexes. The complexes 4 and 5 (100 µM) bind with the minor groove of DNA and cleave double-stranded pBR322 DNA significantly even in the absence of an activator. In the presence of H(2)O(2), they cleave supercoiled DNA via oxidative pathway even at lower concentration (20 µM). Both complexes 4 and 5 inhibit topoisomerase II activity with IC(50) values of 18 and 13. These values suggest that 4 and 5 are potential topoisomerase II inhibitors as compared to some of known inhibitors like novobiocin and etoposide.

Therapeutic effects of oral fluralaner in pet rabbits with severe sarcoptic mange (Sarcoptes scabiei).

Sarcoptic mange is one of the most severe, highly contagious, and fatal ectoparasitic infestations of rabbits. Fluralaner, an isoxazoline class of oral ectoparasiticide, is considered as a very potent acaricide. The present study aimed to evaluate the therapeutic efficacy of oral fluralaner in pet rabbits with severe spontaneous Sarcoptes scabiei infestation. A total of eight un-neutered pet rabbits, tested positive for S. scabiei by microscopy of skin scrapings, were enrolled. Seven rabbits had severe clinical infestation (score 5), while remaining one rabbit had moderate disease clinical signs (score <3). A single oral dose equivalent to 25 mg/kg of fluralaner was administered to each rabbit. On day 14 post-therapy, marked improvements in the skin lesions were observed; severely infested rabbits had a clinical score of 3, while the moderately infested rabbit had a score of 1. However, none of the rabbits tested negative for S. scabiei. On day 30 post-therapy, complete clinical recovery was recorded in all rabbits (Score 0), but, a complete parasitological clearance was not recorded except to the moderately infested rabbit. All rabbits were tested negative for S. scabiei on day 45 post-therapy. Therefore, a single oral dose of fluralaner at a 25 mg/kg was found to be effective in the treatment of severe sarcoptic mange in pet rabbits and no additional topical or systemic medications were needed. Further studies in a larger number of individuals with a bigger spectrum of disease severities (i.e. more moderate/mild) are needed to comprehensively document the safety and efficacy of this drug in mangy rabbits.

Associative Genetic Diversity of RoTat 1.2 VSG in Different Trypanosoma evansi Isolates.

BACKGROUND: Numerous phylogenetic markers have been tested over a period of time for delineating evolutionary history of haemoflagellate-Trypanosoma evansi. PURPOSE: To find out the associative genetic diversity, within the various isolates of T. evansi across the globe, based on RoTat 1.2 VSG gene. METHODS: A total of 5 equine isolates of T. evansi from Northern India were characterized. PCR products were sequenced and sequences were compared with available sequences across India and world. Phylogenetic tree was constructed based on maximum parsimony (MP) method with the tree-bisection-regrafting (TBR) algorithm. RESULTS: Indian isolates formed multiple clades with two haplotypes. The present isolates showed 99.49-100.00% nucleotide homology within themselves. On broader line, Indian isolates were found to be closer to Egyptian isolates than the African counterparts. Few of the Indian isolates showed marked resemblance with a particular Egyptian isolate than with their Indian counter parts. Another remarkable finding is the close association of equine isolates from India with other equine isolates and their clear divergence from isolates of T. evansi affecting other hosts from India and abroad. CONCLUSION: Vast genetic divergence was seen between the isolates suggesting of multiple distinct lineages of T. evansi amongst the Indian livestock. Interestingly, variations in sequences were seen based on the host range of isolates. The findings are very important from molecular evolutionary point of view.

A combined experimental and theoretical investigation of ruthenium(II)-hydrazone complex with DNA: Spectroscopic, nuclease activity, topoisomerase inhibition and molecular docking.

A new Dichlorotetra(4-hydroxy-N'-(pyridin-4-ylmethylene)benzohydrazone)Ru(II) complex 1 has been synthesized and characterized using spectroscopic techniques. The structure of complex 1 has been optimized through ORCA computational programme package using B3LYP functionals. The complex binds efficiently with calf thymus DNA (CT-DNA) as monitored by UV-visible titrations (Kb = 4.8 × 105), ethidium bromide displacement studies (Ksv = 1.39) as well as Circular Dichroism (CD) titrations. The complex intercalates with DNA base pairs. It shows cleavage of supercoiled (SC) DNA into nicked circular (NC) DNA efficiently via oxidative pathway. Complex 1 also inhibits Topoisomerase I (Topo I) relaxation activity at concentration < 20 µM. The molecular docking studies support that Topo I inhibition occur via blocking religation of G11 hydroxyl group.

Characterization of ionic liquid pretreated plant cell wall for improved enzymatic digestibility.

An insight into the properties of cell wall of mustard stalk (MS) pretreated by five ionic liquids (ILs) revealed ILs interaction with cellulose, hemicellulose and lignin components. Differential Scanning Calorimetry (DSC) showed increased pore size coupled with increased population of pores evoked by certain ILs in better facilitating enzymatic accessibility. Interestingly, all the five ILs predominantly increased the propensity of two pore sizes formation; 19 and 198 nm, but remarkable difference in the pore volumes of pretreated MS suggested the supremacy of [OAc]- based ILs, resulting in higher glucose yields. Cellulose I to II transition in pretreated MS was supported by the reduced total crystallinity index (TCI), lateral order index (LOI) values. Strong inverse correlation existed between the said parameters and residual acetyl content with enzymatic hydrolysis (R2 > 0.8). An inverse relationship between hydrogen bond basicity, LOI and TCI suggested it to be a good indicator of IL pretreatment efficiency.

A novel process for extraction of edible oils: Enzyme assisted three phase partitioning (EATPP).

Three phase partitioning (TPP), a technique used in protein purification has been evaluated, for extraction of oil from three different plant sources viz: mango kernel, soybean and rice bran. The process consists of simultaneous addition of t-butanol (1:1,v/v) and ammonium sulphate (w/v) to a crude preparation/slurry. Under optimized condition, the protein appears as an interfacial precipitate between upper t-butanol containing oil and lower aqueous phase. Pretreatment of the slurries with a commercial enzyme preparation of proteases, Protizyme, followed by three phase partitioning resulted in 98%, 86% and 79% (w/w) oil yields in case of soybean, rice bran and mango kernel, respectively. The efficiency of the present technique is comparable to solvent extraction with an added advantage of being less time consuming and using t-butanol which is a safer solvent as compared to n-hexane used in conventional oil extraction process.

Classico-molecular targeting of oligopeptidase B, cysteine protease and variable surface glycoprotein (VSG) genes of Trypanosoma evansi.

Trypanosomosis or Surra can rightly be attributed as the most economically important vector-borne haemoprotozoan disease encountering India. Surra infected chronic cases show almost similar types of signs and symptoms often confusing it with other haemoprotozoan infections, thereby, making it prerequisite for the development of aspecific and sensitive technique for its detection in susceptible animals. Blood microscopy and serology suffers from the hands of lack of sensitivity and specificity thereby leaving molecular detection techniques as one of the promising alternative. Alongside, there is utmost need for exploring of new molecular gene targets for the development of a putative alternative for diagnosis and immunoprophylaxsis. The present communication describes the identification and amplification of oligopeptidase B, cysteine protease and variable surface glycoprotein genes of T. evansi so as to exploit them in future as potential candidates for immune protection and/or molecular detection.

Sexual alteration in antioxidant response and esterase profile in Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks.

The objective of the study was to reveal physiological link between sex specific engorgement pattern and evading mechanism of ticks against oxidative stress as well as acaricides. Quantitative determination of nitric oxide radical scavenging, superoxide dismutase activity and reduced glutathione (GSH) concentrations in salivary gland and gut extracts of male and female Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks established significant variation in antioxidant responses between two sexes of the ticks. Higher activity of these antioxidants and GSH depletion in females clearly indicate stronger antioxidant defense in female ticks which is to combat host mediated oxidative assault during feeding for greater engorgement and reproductive stress. The females are also better equipped with the mechanism of acaricide resistance as evidenced by higher expression of esterases than males in unfed whole tick extracts in current study.

Synthesis, structures, nuclease activity, cytotoxicity, DFT and molecular docking studies of two nitrato bridged homodinuclear (Cu-Cu, Zn-Zn) complexes containing 2,2'-bipyridine and a chalcone derivative.

Nitrato briged dinuclear complexes of type [Cu2(L)2(bpy)2(NO3)](NO3)·4H2O, 1 and [Zn2(L)2(bpy)2(NO3)](NO3)·4H2O, 2 (L=deprotonated form of free ligand LH, [1-(2-hydroxyphenyl)-3-(9-anthracenyl) propenone; bpy=2,2'bipyridine] are synthesized and characterized using a battery of physicochemical techniques and X-ray crystallography. A distorted square pyramidal geometry is assigned to them with N2O3 coordination core around the metal ion. The co-ligand L binds the metal ions through its O,O' atoms in anti-syn mode. The metal centers in complexes 1 and 2 are separated via bridging nitrato group at a distance of 6.073Å and 5.635Å respectively. Their structures and absorption spectra are supported by the computational studies using density functional theory (DFT) and TD-DFT. Both complexes exhibit nuclease activity and cleave supercoiled (form I) DNA. The complex 1 preferentially binds major groove of DNA and follows an oxidative pathway whereas complex 2 binds with minor groove of DNA via hydrolytic pathway. Both complexes inhibit topoisomerase I relaxation activity with IC50 values of 7 and 35µM. Molecular docking studies support the groove binding and topoisomerase I binding of the complexes. The complex 1 showed a significant cytotoxicity against HeLa cell lines (a cervical cancer cell lines) in vitro with IC50 value calculated as 2.9±0.021µM as compared to 28.2±0. 044µΜ for complex 2. Complex 2 induces the cell apoptosis at a later-stage as compared to complex 1. The cell apoptosis and topoisomerase inhibition by complexes enable them to be potential candidates as future anticancer drugs.

Intensification of steam explosion and structural intricacies impacting sugar recovery.

Dilute acid (DA) pretreatment at pilot level failed for cotton stalk (CS) due to the technical issues posed by its inherent nature. Reasonable glucan conversion has been reported via two-stage pretreatment but adds on to the process cost. Proposed herewith is a single-stage steam explosion (SE) process preceded by water extraction resulting in high sugar recovery from CS. Raising the extraction temperature to 80°C increased the glucan conversion from 37.9 to 52.4%. Further improvement up to 68.4% was achieved when DA was incorporated during the room temperature extraction. LC-MS revealed the formation of xylo-oligomers limiting the glucan conversion in proportion to the length of xylo-oligomers. Varying extraction conditions induced structural alterations in biomass after SE evident by compositional analysis, Infrared Spectroscopy, X-Ray Diffraction and Scanning Electron Microscopy. Overall glucose recovery, i.e. 75.8-76.7% with and without DA extraction respectively was achieved.

Ionic liquid pretreatment of biomass for sugars production: Driving factors with a plausible mechanism for higher enzymatic digestibility.

In this study, five ionic liquids (ILs) have been explored for biomass pretreatment for the production of fermentable sugar. We also investigated the driving factors responsible for improved enzymatic digestibility of various ILs treated biomass along with postulating the plausible mechanism thereof. Post pretreatment, mainly two factors impacted the enzymatic digestibility (i) structural deformation (cellulose I to II) along with xylan/lignin removal and (ii) properties of ILs; wherein, K-T parameters, viscosity and surface tension had a direct influence on pretreatment. A systematic investigation of these parameters and their impact on enzymatic digestibility is drawn. [C2mim][OAc] with ß-value 1.32 resulted 97.7% of glucose yield using 10 FPU/g of biomass. A closer insight into the cellulose structural transformation has prompted a plausible mechanism explaining the better digestibility. The impact of these parameters on the digestibility can pave the way to customize the process to make biomass vulnerable to enzymatic attack.

Sex dependent alterations in the protein characterization patterns of Haemonchus contortus.

The aim of the study was to highlight the sex dependent differences in the electrophoretic protein patterns of male and female Haemonchus contortus worms SDS based polyacrylamide gels of both male and female worms were run side by side for comparison. A total of 33 and 35 polypeptides were detected in polyacrylamide gels stained with coomassie brilliant blue R-250, respectively. Besides many of the fundamental homologies in protein profile, some of the polypeptides specific to either sex were also observed. Most of the characteristic polypeptides were of low molecular weight. These polypeptides needs deeper unrevealing regarding the nature of protein, through well planned zymographic studies, so as to ascertain the true nature and/or type of protein involved in those bands. This will help us in better understanding of parasite immunology and sex influenced differences amongst the worm and the possible variations in their pathogenesis contributed thereof, if any.

Pilot scale study on steam explosion and mass balance for higher sugar recovery from rice straw.

Pretreatment of rice straw on pilot scale steam explosion has been attempted to achieve maximum sugar recovery. Three different reaction media viz. water, sulfuric acid and phosphoric acid (0.5%, w/w) were explored for pretreatment by varying operating temperature (160, 180 and 200°C) and reaction time (5 and 10min). Using water and 0.5% SA showed almost similar sugar recovery (∼87%) at 200 and 180°C respectively. However, detailed studies showed that the former caused higher production of oligomeric sugars (13.56g/L) than the later (3.34g/L). Monomeric sugar, followed the reverse trend (7.83 and 11.62g/L respectively). Higher oligomers have a pronounced effect in reducing enzymatic sugar yield as observed in case of water. Mass balance studies for water and SA assisted SE gave total saccharification yield as 81.8% and 77.1% respectively. However, techno-economical viability will have a trade-off between these advantages and disadvantages offered by the pretreatment medium.

The flavonoid derivative 2-(4' Benzyloxyphenyl)-3-hydroxy-chromen-4-one protects against Aß42-induced neurodegeneration in transgenic Drosophila: insights from in silico and in vivo studies.

In the pathogenesis of Alzheimer's disease (AD), it is well established that the self-association of Aß peptides into amyloid fibrils and/or plaque like aggregates causes neurotoxicity. As there is no cure for AD till date, identification of specific compounds that either inhibit the formation of Aß-fibrils or help in the dissolution of already formed amyloid plaques makes an appealing therapeutic and preventive strategy in the development of drugs. In the present study, four synthetic flavonoid derivatives (1, 2, 3 and 4) were examined for docking studies with Amyloid beta (PDB Code: 1IYT) and Amyloid fibril (PDB Code: 2BEG). Of these, compound 1 and 4 were found to be potential inhibitors, as supported by computational molecular docking studies with adequate pharmacokinetic properties. Compound 1 was further tested in vivo in transgenic AD model of Drosophila. The disease causing human Aß42 peptide was expressed in the compound eye by driving UAS-Aß42 with ey-GAL4, which caused severe degeneration in eye tissues ranging from loss of bristles, ommatidial holes to severe ommatidial disruption as revealed by digital camera imaging and scanning electron microscopy. When the Aß42 expressing larvae were grown in medium containing Compound 1, ~70 % rescue of the rough eye phenotype was observed at 75 and 100 µM concentrations. This is further corroborated by significant reduction in amyloid plaques in eye imaginal disks of compound 1 treated larvae as revealed by immuno-confocal imaging studies. Further, rescue of locomotor deficit and improved life span in compound 1 treated Aß flies also confirm the neuroprotective activity of this compound. Thus, our results support the neuroprotective efficacy of compound 1 in preventing Aß42-induced neurotoxicity in vivo and identify it as a future therapeutic agent against AD.

Protein-coated microcrystals of Pseudomonas aeruginosa PseA lipase.

Highly active Pseudomonas aeruginosa lipase protein-coated microcrystals (PAL PCMC) have been prepared by immobilization of protein onto K(2)SO(4) as excipient solid support carrier and n-propanol as precipitating solvent. Transmission electron micrographs confirmed the formation of PAL PCMC. These PCMC were found to be a catalytically more active and stable preparation for p-nitrophenyl palmitate hydrolysis in n-heptane, compared to free lipase. The V (max), K (m), and temperature optimum for PAL PCMC increased from 0.49 to 5.66 nmol min(-1) mg(-1), 589 to 679.8 mmol, and 40 degrees C to 45 degrees C, respectively. These were thermally more stable with 4.65, 2.56, and 1.24-fold improvement in half lives at 45 degrees C, 55 degrees C, and 60 degrees C compared to free P. aeruginosa PseA lipase. Their catalytic efficiency was enhanced by tenfold over that of free enzyme. PAL PCMC offer a simple and effective technique for obtaining stable and efficient lipase preparation for biocatalysis in nonaqueous medium.