Enzymatic Esterification of Indole-3-acetic Acid to myo-Inositol and Glucose

Incubation of mature sweet corn kernels of Zea mays in dilute solutions of ¹⁴C-labeled indole-3-acetic acid leads to the formation of ¹⁴C-labeled esters of myo-inositol, glucose, and glucans. Utilizing this knowledge it was found that an enzyme preparation from immature sweet corn kernels of Zea mays catalyzed the CoA- and ATP-dependent esterification of indole-3-acetic acid to myo-inositol and glucose. The esters formed were 2-O-(indole-3-acetyl)-myo-inositol, 1-dl-1-O-(indole-3-acetyl)-myo-inositol, di-O-(indole-3-acetyl)-myo-inositol, tri-O-(indole-3-acetyl)-myo-inositol, 2-O-(indole-3-acetyl)-d-glucopyranose, 4-O-(indole-3-acetyl)-d-glucopyranose and 6-O-(indole-3-acetyl)-d-glycopyranose. An assay system was developed for measuring esterification of ¹⁴C-labeled indole-3-acetic acid by ammonolysis of the esters followed by isolation and counting the radioactive indole-3-acetamide.     

Isolation and Characterization of a Novel p-Coumaric Acid Ester of myo-Inositol from Needles of Taxus baccata

A novel derivative of myo-inositol was isolated from the needles of Taxus baccata and identified as p-coumaric acid ester. By means of its levorotatory optical activity and formation of a borate complex, the compound was designated tentatively as a 1l-4-p-coumaroyl-myo-inositol. Chemotaxonomic investigations have shown this ester to be present in needles of 14 out of 34 tested species of gymnosperms. When (14)CO(2) or (14)C-myo-inositol were fed to young needles of T. baccata, there was a rapid incorporation of label in the p-coumaroyl ester, and a pulse-chase experiment indicated a substantial turnover of this compound over a 4-month period.     

Cofactor Recycling for Selective Enzymatic Biotransformation of Cinnamaldehyde to Cinnamyl Alcohol

The enzymatic, selective hydrogenation of cinnamaldehyde to cinnamyl alcohol is reported here. Yeast alcohol dehydrogenase was used in a substrate-coupled process with cofactor recycling. Both 100% selectivity and aldehyde conversion were achieved within 3 h. The reaction took place under very mild conditions, in the absence of toxic organic solvent. The overall process proved inexpensive and deserves further optimization studies in order to evaluate industrial applications.     

毛栓菌漆酶的分离纯化及酶学性质研究

对毛栓菌产漆酶的分离、纯化及酶学性质进行研究。粗酶液经硫酸铵盐析、透析、DEAE-Sepharose柱层析,得到2种漆酶同工酶LacA和LacB。LacA和LacB回收率分别为17.1%和2.74%。SDS-PAGE电泳测得2漆酶的分子量分别为54.6 ku和7.7 ku;LacA和LacB最适作用温度分别为50℃和60℃;最适反应pH值分别为4.5和4.0;Cu2＋、Mg2＋对LacA有激活作用,对LacB影响不大;Ag＋对LacA和LacB表现为完全抑制;Fe3＋对LacA和LacB有一定的抑制作用;Ca2＋、Mn2＋、K＋、Na＋、Zn2＋对LacA和LacB影响不大。DTT、EDTA、DMSO、SDS对酶均有不同程度的抑制作用,且随其浓度的升高抑制作用增强。     

Purification of myo-Inositol 1-Phosphate Synthase from Rice Cell Culture by Affinity Chromatography

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) is the enzyme which catalyzes the synthesis of the precursor for the myo-inositol oxidation pathway. Rice callus grown in suspension culture provides a good source of plant enzyme. Use has been made of a noncompetitive inhibitor to prepare an affinity column for this enzyme. With this column, the enzyme from rice callus has been purified 1500-fold in a single step, about 9000-fold over-all, to a specific activity of 0.078 units per milligram of protein. This is an order of magnitude greater than previous purifications of the plant enzyme.     

Metabolism of myo-Inositol by Germinating Lilium longiflorum Pollen

Lilium Iongiflorum pollen tubes absorbed myo-[2-³H]inositol produced labeled metabolites which were separated into acid-soluble and -insoluble fractions. The soluble fraction contained labeled myo-inositol, d-glucuronic acid, myo-inositol 1-phosphate, and at least three other unidentified compounds. The acid-insoluble fraction contained considerable chloroformsoluble radioactivity and a labeled residue. Labeled myo-inositol was also absorbed by germinating pollen prior to the time of pollen tube initiation; however, there was a marked reduction in amounts of myo-inositol 1-phosphate and glucuronic acid produced by this pollen in comparison with growing pollen tubes.     

沼泽红假单胞菌粗酶液生物转化柚皮苷

目的初步探索沼泽红假单胞菌粗酶液生物转化柚皮苷的特点。方法利用高效液相色谱技术,以柚皮苷的降解率为指标,考察不同温度、pH、底物浓度和培养时间对粗酶液降解柚皮苷的影响。在适宜转化条件下,探索底物和产物的含量变化。结果最适转化条件:40℃~50℃,pH 7.0~8.0,最大有效转化底物浓度750μg/mL,15 h时柚皮苷降解率较高为55.31%。在最适转化条件下,底物浓度500μg/mL,培养至17 h柚皮苷被降解完全,柚皮素浓度达到最大值204μg/mL,转化率为85.39%;10~19 h,转化率均大于80.00%,13 h达到最大值94.83%。结论本研究首次发现沼泽红假单胞菌粗酶液能够生物转化柚皮苷,并阐明了不同培养条件对柚皮苷降解的影响,以及转化过程中物质含量的变化。     

Biotransformation of Saponins by Endophytes Isolated from Panax notoginseng

The biotransformation of the major saponins in Panax notoginseng, including the ginsenosides Rg1, Rh1, Rb1, and Re, by endophytes isolated from P. notoginseng was studied. One hundred and thirty‐six endophytes were isolated and screened for their biotransformational abilities. The results showed that five of the tested endophytes were able to transform these saponins. These five strains were identified based on their ITS or 16S rDNA sequences, which revealed that they belonged to the genera Fusarium, Nodulisporium, Brevundimonas, and Bacillus genera. Ten transformed products were isolated and identified, including a new compound 6‐O‐[α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐glucopyranosyl]‐20‐O‐β‐D ‐glucopyranosyldammarane‐3,6,12,20,24,25‐hexaol ( 3 ), and nine known compounds, compound K ( 1 ), ginsenoside F2 ( 2 ), vinaginsenoside R13 ( 4 ), vinaginsenoside R22 ( 5 ), pseudo‐ginsenoside RT4 ( 6 ), (20S)‐protopanaxatriol ( 7 ), ginsenoside Rg1 ( 8 ), vinaginsenoside R15 ( 9 ), and (20S)‐3‐O‐β‐D ‐glucopyranosyl‐6‐O‐β‐D ‐glucopyranosylprotopanaxatriol ( 10 ). This is the first study on the biotransformation of chemical components in P. notoginseng by endophytes isolated from the same plant.     

猪骨胶原蛋白酶解物中血管紧张素转换酶抑制剂的提纯

将猪骨胶原蛋白粗提物用胰蛋白酶水解,经阳离子交换树脂层析,ＳｅｐｈａｄｅｘＧ－２５柱凝胶过滤,以及数次反相高效液相层析,最终获得一具有抑制血管紧张素转换酶（ＡＣＥ）活性的单一峰值的多肽。其氨基酸组成为Ｉｌｅ,Ｈｉｓ,Ｓｅｒ,Ｇｌｙ,Ａｌａ,Ｐｒｏ,Ｔｙｒ,Ｌｅｕ,Ａｓｐ．以Ｈｉｐ－Ｇｌｙ－Ｇｌｙ为底物,在ｐＨ为７．１的条件下,此肽对猪肺ＡＣＥ的Ｉ＿（５０）值为２６μｍｏｌ／Ｌ。     

Enzymatic Isolation of Protoplasts from Fern Protonemal Cells Stainable with a Fluorescent Brightener

Protoplasts were successfully isolated for the first time fromthe filamentous protonemal cells of ferns after the cells werecultured in contact with both air and medium. Sterilized sporesof Adiantum capillus-veneris and Pteris vittata were inoculatedon a piece of nylon mesh (40- {diaeresis}m mesh) placed on amat of polyester fibers which was soaked in liquid culture medium,and the spores were illuminated from above with continuous redlight. Protonemal cells, exposed to the air during this procedure,could be stained with Calcofluor White, a dye that binds tocell walls. Protoplasts were easily isolated from these protonemalcells by digestion of the cell wall with cellulase and pectinase.A total of 0.8–1.9 x 10⁴ and 0.6–2.0 x 10⁴ protoplastswere obtained from protonemata that originated from 10 mg ofdry spores of Adiantum and of Pteris respectively. Viability,as judged by staining with fluorescein diacetate was more than90% for both species. Staining with 4'6-diamidino-2-phenylindole(DAPI) revealed that about half of the protoplasts of both speciescontained a nucleus. (Received May 22, 1989; Accepted September 5, 1989)     

Biotransformation of arsenobetaine by microorganisms from the human gastrointestinal tract

Abstract

The consumption of fish and shellfish is a major route of human exposure to arsenic (As), because they contain relatively large concentrations of organoarsenicals, in particular arsenobetaine (AB). AB is considered non-toxic because of its rapid excretion from the human body. However, previous studies on human metabolism and excretion of AB have used the compound in solution rather than considering the effects that occur during the digestion of food in the gastrointestinal tract. In this preliminary study, we used microcosms inoculated with human faecal matter to investigate the aerobic and anaerobic degradation of AB by microorganisms associated with the large intestine. Samples were recovered over 30 days, centrifuged, filtered and the supernatant analysed for total As content and As speciation, using ICP–MS and HPLC–ICP–MS respectively. After 7 days the total As in the supernatants from the aerobic experiment fell to a minimum of 65% of the total added, recovering to 15% less than added after 30 days. By using anion and cation exchange chromatography coupled to ICP–MS detection, arsenobetaine (AB), dimethylarsinic acid (DMA), dimethylarsinoylacetic acid (DMAA) and trimethylarsine oxide (TMAO) were identified as degradation products. Results from the aerobic system showed that after 7 days incubation the AB had been degraded to DMA, DMAA and TMAO and after 30 days the degraded AB reappeared in the samples. The results for the anaerobic system showed no degradation of AB over the 30 day course of the experiment. These findings demonstrate for the first time that biocatalytic capability for AB degradation exists within the human gastrointestinal tract.     

Biotransformation of cycloalkanones by microorganisms

Summary Washed-cell suspensions of cyclopentanol-grownPseudomonas sp. NCIB 9872 were shown to preferentially biotransform norbornanone to an equivalent lactone of potential use in the synthesis of prostanoid analogues. Conditions to optimise the yield of product, including incubation at pH 7.1–7.35 and at substrate concentrations <15 mM, have been established.     

Biotransformation of N-acetylphenothiazine by fungi

Cultures of the fungi Aspergillus niger, Cunninghamella verticillata, and Penicillium simplicissimum, grown in a sucrose/peptone medium, transformed N-acetylphenothiazine to N-acetylphenothiazine sulfoxide (from 13% to 28% of the total) and phenothiazine sulfoxide (from 5% to 27%). Phenothiazin-3-one (4%) and phenothiazine N-glucoside (4%) were also produced by C. verticillata. The probable intermediate, phenothiazine, was detected only in cultures of P. simplicissimum (6%). Received: 15 January 1999 / Received revision: 7 May 1999 / Accepted: 21 May 1999     

Biotransformation of albendazole by fungi

Twenty-five fungal cultures were screened for their ability to transform the anthelmintic drug albendazole. A filamentous fungi Cunninghamella blakesleeana transformed albendazole to three metabolites in significant quantities. The transformation of albendazole was identified by HPLC. Based on the LC-MS-MS data, two metabolites were predicted to be albendazole sulfoxide and albendazole sulfone, the major mammalian metabolites reported previously. A new N-methylated metabolite of albendazole sulfoxide was also produced, where the methylation took place on the N-atom of the imidazole ring system. A temperature of 30°C, pH of 8 and high substrate concentrations produced highest transformation of albendazole. Among the various concentrations studied, 2% w/v of glucose produced highest transformation. The results reveal that the microbial model can be used to produce large quantities of mammalian metabolites.     

Biotransformation of nitriles by rhodococci

Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.     

myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on phytate

For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.     

Biotransformation of flavone by CYP105P2 from Streptomyces peucetius

Biocatalytic transfer of oxygen in isolated cytochrome P450 or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. Cytochrome P450 CYP105P2 was isolated from Streptomyces peucetius that showed a high degree of amino acid identity with hydroxylases. Previously performed homology modeling, and subsequent docking of the model with flavone, displayed a reasonable docked structure. Therefore, in this study, in a pursuit to hydroxylate the flavone ring, CYP105P2 was co-expressed in a two-vector system with putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida for efficient electron transport. HPLC analysis of the isolated product, together with LCMS analysis, showed a monohydroxylated flavone, which was further established by subsequent ESI/MS-MS. A successful 10.35% yield was achieved with the whole-cell bioconversion reaction in Escherichia coli. We verified that CYP105P2 is a potential bacterial hydroxylase.     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.