Lipase-mediated epoxidation of α-pinene

This work describes the lipase-mediated synthesis of α-pinene oxide at ambient temperature. The immobilized lipase from Candida antarctica (Novozyme 435) is used to generate peroxyoctanoic acid directly from octanoic acid and hydrogen peroxide. The peroxy acid formed is then applied for in situ oxidation of α-pinene. High conversion of α-pinene to α-pinene oxide (approximately 70%) was achieved when using a two-phase system of toluene and water. Various parameters affecting the conversion of α-pinene to α-pinene oxide were studied.     

Thermophilic biofiltration of methanol and α-pinene

Biofiltration systems utilizing thermophilic (55C) bacteria were constructed and tested for the removal of methanol and α-pinene — two important volatile organic compounds (VOCs) in the forest products industry. Thermophilic bacterial mixtures that can degrade both methanol and α-pinene were obtained via enrichment techniques. Two bench-scale thermophilic biofiltration systems (1085 and 1824 cm³) were used to examine compound removals at different residence times, with influent concentrations of 110 ppmv methanol and 15 ppmv α-pinene. At a residence time of 10.85 min, the smaller system had removal efficiencies of >98% for methanol, but only 23% for α-pinene. The larger system was operated with the same parameters to evaluate residence time and surfactant effects on compound removals. At a residence time of 18.24 min, both methanol and α-pinene removal rates were ≥95%. However, α-pinene removal dropped to 26% at a residence time of 6.08 min; methanol removal was not affected. Subsequent addition of a surfactant mixture increased α-pinene removal to 94% at the shortest residence time. No residual α-pinene was detected with the support medium Celite R-635, indicating that the surfactant may increase mass transfer of α-pinene. Journal of Industrial Microbiology & Biotechnology (2001) 26, 127–133. Received 06 June 2000/ Accepted in revised form 09 November 2000     

Microbial α-mannosidases

Microbial α-mannosidases are used in the analysis of glycopeptides and the developmental regulation of lysosomal enzymes. This survey presents comparison of properties of this high molecular weight, oligomeric protein from a number of microbial sources.     

Microbial production and chemical transformation of poly-γ-glutamate

Poly-γ-glutamate (PGA), a novel polyamide material with industrial applications, possesses a nylon-like backbone, is structurally similar to polyacrylic acid, is biodegradable and is safe for human consumption. PGA is frequently found in the mucilage of natto, a Japanese traditional fermented food. To date, three different types of PGA, namely a homo polymer of d-glutamate (D-PGA), a homo polymer of l-glutamate (L-PGA), and a random copolymer consisting of d- and l-glutamate (DL-PGA), are known. This review will detail the occurrence and physiology of PGA. The proposed reaction mechanism of PGA synthesis including its localization and the structure of the involved enzyme, PGA synthetase, are described. The occurrence of multiple carboxyl residues in PGA likely plays a role in its relative unsuitability for the development of bio-nylon plastics and thus, establishment of an efficient PGA-reforming strategy is of great importance. Aside from the potential applications of PGA proposed to date, a new technique for chemical transformation of PGA is also discussed. Finally, some techniques for PGA and its derivatives in advanced material technology are presented.     

Microbial transformation ofβ-sitosterol byNocardia sp. M 29

Summary The degradation and conversion of -sitosterol to C-17-ketosteroids byNocardia sp. M 29 was studied. Maximal enzymatic activity was found after 24 h of incubation. Although the key enzymes involved in the decomposition of -sitosterol were inducible, no separate induction of side chain hydroxylase or 9-hydroxylase was possible. Inhibition of the steroid ring cleaving enzyme by ,-dipyridyl resulted in low yields of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione (total yield 22%). Addition of lipophilic organic adsorbents (Amberlite XAD-2 and XAD-4) stimulated the 1,4-androstadiene-3,17-dione formation (maximal 50% within 120 h of incubation). Furthermore, 3-oxo-23,24-dinor-1,4-choladienic acid and 3-oxo-23,24-dinor-1,4-choladienic acid methyl ester were accumulated in the presence of Amberlite XAD-2 in moderate yields (total 11%).     

Microbial Oxidation of α-Methylstyrene and β-Methylstyrene

An unidentified bacterial strain S107B1, isolated from soil by use of isopropylbenzene as a carbon source, was shown to bring about oxidation of α-methylstyrene and β-methylstyrene,

One of the oxidation products produced from α-methylstyrene was identified as the new compound, (—)-cis-23-dihydroxy-1-isopropenyl-6-cyclohexene.

The same strain S107B1 also oxidized β-methylstyrene and produced 3-phenylpropionaldehyde and benzoic acid.

From these results, the existence of reductive step for the aerobic degradation of these aromatic hydrocarbons by this strain was made clear. The initial attack on these aromatic hydrocarbons and a cyclohexenediol compound formed from α-methylstyrene were discussed.     

Biotransformation of α- and β-pinene into flavor compounds

Products that bear the label “natural” have gained more attention in the marketplace. In this approach, the production of aroma compounds through biotransformation or bioconversion has been receiving more incentives in economic and research fields. Among the substrates used in these processes, terpenes can be highlighted for their versatility and low cost; some examples are limonene, α-pinene, and β-pinene. This work focused on the biotransformation of the two bicyclic monoterpenes, α-pinene and β-pinene; the use of different biocatalysts; the products obtained; and the conditions employed in the process.

     

Microbial transformation of steroids XVIII. Dehydrogenation of cortisone in position 1—2

У 248 ш0442;аммов различн044B;х родов (Fusarium, Actinomyces, Proactinomyces, Nocardia, Mycobacterium) автор044B; иисслeдо вали способность дeгидрировать кортизон в 043F;оложeнии 1–2 стeроидного скe043B;eта. у штаммов, у которых была доказана эта способность к дeгидрированию, He было найдeно различий в качeствe возника ющих мeтаболитов, однако скорость прeв ращeния у отдeльных штаммов He была одинаковой. Самоe быстроe тeчeниe прeвращeния наблюдалось у штамма Mycobacterium flavum № 390. У этого штамма мы исслeдовали поэтому влияниe на процeсс прeвv0440;ащeния — воз раста, нарастания культуры, рН срeды и тeмпeратуры фeрмeнтации. На качe ство возникающих мeтаболитов Нe удалось воздeй ствовать ни одним из этих факторов. При прeвращeнии кортизона при помощи указанного штамма в качeствe пeрвого продукта возникаeт прeднизон, который однко далee восстанавливаeтся в по043B;ожeнии 20 в соотвeтствующee 20 β-оксиосоизводноe. рАзвитиE БАктЕрио цидного дЕйствия нΑ грΑм-отрицΑтΕльныΕ оргΑнизмы Β сыΒороткΑх молодых животных Я. Шmе? я. коска Α. Лан Исследовали бакт ериоцидную актикн ость сыBороток, получен ных от поросят, вскормленных в ссерильных условиях, без материнского молозива. Сыворотки зтих поросят содержат комплемент, но не содержат ни малейших следов антител и не обладают бакт ериоцидным действиен по отношению к микробам Εсе са со и eаmо еа, находящимся в e-фазеΒ сыворотках стер ильных молодых Животных, искус ственно заселенных определенным типом микроба Ε. со, бактериоцидная активность была обнаружена уже на 4-7-й день после введения микробов. Зта бактериоцидная активность 0ка зывается спепифич еской только по отношению к штамму, которым поросята были заселены.     

Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

Microbial metabolism of α-mangostin isolated from Garcinia mangostana L

α-Mangostin (1), a prenylated xanthone isolated from the fruit hull of Garcinia mangostana L., was individually metabolized by two fungi, Colletotrichum gloeosporioides (EYL131) and Neosartorya spathulata (EYR042), repectively. Incubation of 1 with C. gloeosporioides (EYL131) gave four metabolites which were identified as mangostin 3-sulfate (2), mangostanin 6-sulfate (3), 17,18-dihydroxymangostanin 6-sulfate (4)and isomangostanin 3-sulfate (5). Compound 2 was also formed by incubation with N. spathulata (EYR042). The structures of the isolated compounds were elucidated by spectroscopic data analysis. Of the isolated metabolites, 2 exhibited significant anti-mycobacterial activity against Mycobacterium tuberculosis.     

Microbial Degradation of α-Picolinic Acid, 2-Pyridinecarboxylic Acid

The biological effects of raw winged bean seeds were investigated with feeding experiments on rats, and the effects of lectin (phytohemagglutinin) present in the seeds are discussed. Administration of a 30% raw winged bean diet caused strong growth depression in young rats, and led to death within 10 ~ 20 days, inducing severe damage to the small intestine of the rats. Significant morphological changes of the intestinal mucosa were observed with a microscopic investigation. As the lethal effect was eliminated by autoclaving but not removed with supplementation of 0.5% l-methionine to the raw winged bean diet, the lectin was assumed to be closely related to the deleterious effects of raw winged bean. In vitro and in vivo digestion tests of the lectin revealed that the winged bean lectin had resistance to peptic, pancreatic and membrane digestions. The hemagglutinating activity was also detected in the intestinal mucosa and faeces from rats ingesting the raw winged bean or its purified lectin. The binding action of lection to mucosal epitheliums of the gastrointestinal tract is suggested to be the initial step of the deleterious effects induced by the winged bean lectin.     

Microbial Reduction of 1,2-Diketones to Optically Active α-Hydroxyketones

We have attempted to develop an intraoral method which can measure the textural changes in foodstuffs during chewing by using electromyography (EMG). Forty-three foodstuffs with variable textural attributes were used.

Total chewing energy for these foodstuffs during chewing varied from 3 to 108 for the masseter muscle and 13 to 154 for the digastric muscle, respectively. Large differences in total chewing energy could be observed by EMG among the foodstuffs. The chewing energy for many foodstuffs revealed distinct differences throughout the chewing process. Foodstuffs could be categorized into six groups according to the changing patterns of chewing energy. EMG data and the number of strokes were influenced by masticatory index and salivary flow rate.     

Microbial transformation of 3β-acetoxypregna-5,16-diene-20-one by Penicillium citrinum

The biotransformation of 3β-acetoxypregna-5,16-diene-20-one (1) by using a filamentous fungus Penicillium citrinum resulted in the production of four metabolites 2–5. The structures of these compounds were elucidated by different spectroscopic analysis (1D- and 2D-NMR) and HR-ESI-MS as 3β,7β-dihydroxy-pregn-5,16(17)-dien-20-one (2), 3β-hydroxy-7α-methoxy-pregn-5,16(17)-dien-20-one (3), 3β,7β,11α-trihydroxy-pregn-5,16(17)-dien-20-one (4), and a known 3β,7α-dihydroxy-pregn-5,16(17)-dien-20-one (5). The 7-O-methylation is a novel reaction in the field of microbial transformation of pregnane steroids.     

Pheromones of Dendroctonus: Origin of α-pinene oxidation products present in emergent adults

Experiments showed that larvae and adults of the bark beetles Dendroctonus terebrans and Dendroctonus frontalis are capable of metabolizing α-pinene, a component of the oleoresin of their host Pinus taeda, to produce large quantities of oxidation products such as trans-verbenol, whereas the pupae do not. The results suggest that the pupae conjugate some form of the terpene molecule with an unknown compound and this conjugate is later metabolized by the young adult to yield the previously identified oxidation products found in emergent beetles. Only adult males of D. frontalis produced large quantities of the ketone verbenone. This compound was not detectable in the hindguts until after the adult maturation period and its production by emergent males could be related to the exposure of the pupae to α-pinene vapours. D. frontalis males are also capable of producing verbenone from α-pinene taken up in the adult stage. It is suggested that the production of verbenone by the males represents a specialization in the evolution of chemical communication in bark beetles. On the basis of this and earlier work, it is considered likely that other terpenes are metabolized in the same manner and that the same or a very similar system of terpene metabolism exists in other Dendroctonus species and closely related genera.     

Microbial transformation of 18β-glycyrrhetinic acid by Sphingomonas paucimobilis strain G5

The effects of the oxygenase inhibitors, 1-aminobenzotriazole (ABT), ketoconazole, metyrapone and proadifen, on the metabolism of 18-glycyrrhetinic acid (18-GRA) in Sphingomonas paucimobilis strain G5 were investigated. Strain G5 transformed 18-GRA into a major new metabolite (M-D) in the presence of 1 mM ABT or metyrapone. M-D was purified and identified as 3-hydroxy-11-oxo-olean-12-en-24,30-dioic acid by NMR and MS. Based on the structure of M-D, we propose the metabolic pathway of 18-GRA in strain G5.     

Potential α-glucosidase inhibitors from thermal transformation of (+)-catechin

Thermal transformation of the (+)-catechin (1) with heating processing afforded a new oxidation product, gambiriin D (2), along with catechin [6′–8]-catechin (3), and (+)-epicatechin (4). The structure of a new catechin dimer with C–C linkage was determined on the basis of spectroscopic data interpretation. The catechin dimers 2 and 3 exhibited significantly improved inhibitory activities against α-glucosidase, with IC₅₀ values of 0.16 ± 0.2 and 0.14 ± 0.2 μM, respectively, when compared to parent (+)-catechin. Kinetic analysis showed that the two effective compounds 2 and 3 have noncompetitive modes of action.     

Microbial transformation of β-lapachone to its glycosides by Cunninghamella elegans ATCC 10028b

β-lapachone (1) has entered phases I and II clinical trials for the treatment of solid tumors and the therapeutic efficacy of β-lapachone is closely related to its metabolic process. In order to contribute to a better understanding of human metabolism of β-lapachone, Cunninghamella elegans ATCC 10028b was used as a microbial model of mammalian metabolism to biotransform β-lapachone and two new glycosylated derivatives were produced. The chemical structures were elucidated as 6-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-5-O-β-d-glucopyranoside (2) and 5-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-6-O-β-d-glucopyranoside (3) by ¹H NMR, ¹³C NMR, HMBC, HMQC, COSY and HRMS analyses. The major derivative (3) displayed a lower activity against breast cancer cell line SKBR-3 (IC₅₀ = 312.5 μM) than β-lapachone (IC₅₀ = 5.6 μM), but did not show cytotoxicity against normal fibroblasts cell line GM07492-A, whereas β-lapachone was highly toxic (IC₅₀ = 7.25 μM). These metabolites were reported here for the first time and are similar to those that occur in phase II of human metabolism     

A Study on the Process of Lipase-catalyzed Synthesis of α-pinene Oxide in Organic Solvents

The synthesis of α-pinene oxide was studied in a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene. The peroxycarboxylic acid formed was then used in situ for the oxidation of α-pinene to the corresponding epoxide. When hydrogen peroxide was added in the reaction mixture gradually over 6 h, conversions increased up to 31.6%. Initial rates of α-pinene oxidation increased from 85 to 708 mmol L^?1 h^?1 when the amount of H₂O₂ increased from 5 to 60 mmol. When the lipase was exposed to 75 mmol H₂O₂ for 0.5 h before its addition in the reaction mixture, its activity decreased to about 50%. The reusability of lipase was studied in five reaction cycles and was found to depend on the concentration of the hydrogen peroxide used.     

New Anthracycline Antibiotic CG12 Obtained by Microbial Glycosidation of α-Citromycinone

We studied the effects of peptidylarginine deiminase, a Ca²⁺-dependent protein-modulating enzyme that catalyzes the deimination of arginyl residues in protein on two major trypsin inhibitors in mouse plasma. One of these inhibitors was α-1-antitrypsin and the other was a recently characterized trypsin inhibitor termed contrapsin (H. Takahara and H. Sinohara, J. Biol. Chem., 257, 2438 (1982)). The enzyme abolished the trypsin-inhibiting activity of contrapsin in a process that was pseudo-first order with the rate dependent on enzyme concentration (second order rate constant = 3.4 × 10³ m^-1 · s^-1), but no detectable changes in the activity was noted for α-1-antitrypsin. Millimolar Ca²⁺ and dithiothreitol were absolutely required for the inactivation of contrapsin by peptidylarginine deiminase. Although no significant alterations of the charge and the size in modified contrapsin were observed, the modified inhibitor indicated that 1 arginyl residue was converted to a citrullyl residue. Modified contrapsin whose anti-tryptic activity was lost inhibited chymotrypsin much more strongly than the native inhibitor. These data suggest that a vital amino acid residue for the anti-tryptic activity of mouse contrapsin is an arginyl residue and the conversion of this arginyl residue to a citrullyl residue causes the functional changes of the inhibitor.     

Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate

With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.