Biosorption of uranium by immobilized cells of Rhodotorula glutinis

Biosorption of uranium ions from diluted solution (≤40 mg L^?1) onto immobilized cells of Rhodotorula glutinis was investigated in a batch system. Equilibrium, kinetic and thermodynamic studies were conducted by considering the effect of initial uranium concentration, contact time and temperature. Non-linear forms of Langmuir, Freundlich and Sips isotherm models were used to fit the equilibrium data, Sips model was designated as the best one. Kinetic data were simulated by non-linear pseudo-first-order, pseudo-second-order and intra-particle diffusion equations. Pseudo-first-order kinetic equation described the experimental data better than pseudo-second-order equation and intra-particle diffusion equation can fit the kinetic data with two independent curves. Thermodynamic parameters, including ?H ⁰, ?G ⁰ and ?S ⁰, were evaluated, the sorption process was determined to be spontaneous and endothermic. Uranium sorption from pure uranium solutions and uranium pit wastewater by immobilized biomass and blank beads, as well as the regeneration results indicated that immobilized R. glutinis can be use to recovery uranium from uranium pit wastewater.     

一个具有相反光学选择性的新颖海洋 GDSL脂肪酶MT6用于（S）-1-苯基乙醇制备

1-苯乙醇是一种重要的手性药物中间体,并且(S)-1-苯乙醇和(R)-1-苯乙醇均具有应用价值.怎样获得光学醇的1-苯乙醇是药物合成中的重要问题.传统的化学合成手段不仅反应过程复杂,而且反应条件剧烈,对环境污染严重,因此生物催化方法越来越受到重视.脂肪酶和酯酶以其出色的立体选择性和温和的反应条件而被广泛用于手性药物的拆分制备.但是之前的一些研究发现脂肪酶和酯酶大都对(R)-1-苯乙醇及其衍生物有选择性,而我们发现并鉴定的脂肪酶 MT6的立体选择性则与这些脂肪酶/酯酶完全相反,具体体现在以下两个方面：(1) MT6能够特异地催化(S)-1-苯乙醇和乙酸异丙烯酯的转酯反应,生成(R)-1-苯乙醇;(2) MT6能够选择性地水解(S)-乙酸苏合香酯,生成(S)-1-苯乙醇.可见,利用 MT6催化的转酯反应和水解反应可以巧妙地进行(S)-1-苯乙醇和(R)-1-苯乙醇的制备. MT6来源于深海放线菌Marinactinospora thermotolerans SCSIO 00652,属于 GDSL家族脂肪酶第 II类群,这一类群的脂肪酶绝大多数来自微生物.有关 GDSL家族脂肪酶在手性拆分中的应用研究非常少.我们之前报道了 MT6的克隆、表达、纯化及转酯拆分反应,本文重点考察了 MT6通过水解反应制备(S)-1-苯乙醇的条件,优化了酶促水解拆分反应温度、有机共溶剂、pH、离子强度、酶用量、底物浓度、反应时间以及底物侧链长度等参数.研究发现,在反应体系中加入一定量的有机共溶剂能够大大提高产物(S)-1-苯乙醇的光学纯度,其中添加二氯甲烷获得的结果最为理想,可以将产物光学纯度从43%提高到89%,E值从2.84提高至22.82.经过优化,最佳反应温度为40°C,共溶剂二氯甲烷浓度为5%(体积分数),反应缓冲液为0.1 mol/L Tris-HCl (pH =7.0),酶用量为150 mg/mL,底物为15 mmol/L乙酸苏合香酯,反应时间控制在12 h.在此条件下,制备的(S)-1-苯乙醇的光学纯度可达97%,转化率可达28.5%,E值为95.9.此外,还比较了侧链长度不同的1-苯基乙醇酯对水解反应的影响,结果表明1-苯基乙醇酯的侧链长度可极大影响光学选择性和产率.在反应条件相同时, MT6催化侧链长度为4个碳的丁酸-1-苯乙酯水解,生成(S)-1-苯乙醇的光学纯度仅为50%.利用 AutoDock软件进行分子对接,结果显示长侧链的1-苯基乙醇酯离活性中心 His230的咪唑基较远,可能是导致酶立体选择性低的重要原因.值得注意的是,海洋微生物来源的 GDSL脂肪酶 MT6在水解反应和转酯反应中均表现出与一些已知脂肪酶/酯酶相反的立体选择性,因而具备进一步开发和应用价值.所制备的(S)-1-苯乙醇的光学纯度为97%,可以通过和转酯反应相结合的方式进一步提高产物的光学纯度和转化率.     

Lipase immobilized on HOOC-MCF： A highly enantioselective catalyst for transesterification resolution of （R,S）-1-phenylethanol

Pseudomonas cepacia lipase (PSL) immobilized on the carboxyl-functionalized meso-cellular foams (HOOC-MCF) was used for the transesterification resolution of (R,S)-1-phenylethanol in organic solvent.The results showed that the ee value of (S)-1- phenylethanol and (R)-1-phenylethyl acetate reached 99% with 50% conversion of 1-phenylethanol using toluene as solvent. Furthermore,it was found that PSL/HOOC-MCF exhibited high enantioselectivity in organic solvent with log P≤2 such as toluene and hexane.     

Continuous production of butanol with immobilized cells ofClostridium acetobutylicum

Spores ofClostridium acetobutylicum were immobilized in calcium alginate. An active gel preparation was obtained after outgrowth of the spores to vegetative cells within the gel matrix. A 100 mL column containing the immobilized cells was used for continuous production. At steady-state conditions the productivity of butanol was 67 g/L reactor volume/day.     

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×10⁵ Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.     

Highly efficient double enantioselection by lipase-catalyzed transesterification of (R,S)-carboxylic acid vinyl esters with (RS)-1-phenylethanol

Transesterification of the title compounds using lipase B from Candida antarctica in toluene afforded the corresponding esters in good to excellent diastereomeric excess. (R)-2-Phenylpropionic acid-(R)-1-phenethyl ester 4 was isolated in 45% yield and 64% de after 2.5 h, whereas (R)-2-phenylbutyric acid-(R)-1-phenethyl ester 5 was obtained in 40% yield and 56% de after 35 h. A single recrystallization from n-hexane gave 4 with 98% de. In all reactions CAL-B showed excellent enantioselectivity (E >100) toward (RS)-1-phenylethanol and moderate enantioselectivity (E∼10) toward both carboxylic acid vinyl esters.     

A new strategy for lipid production by mix cultivation of Spirulina platensis and Rhodotorula glutinis

Mix cultivation of microalgae (Spirulina platensis) and yeast (Rhodotorula glutinis) for lipid production was studied. Mixing cultivation of the two microorganisms significantly increased the accumulation of total biomass and total lipid yield. Dissolved oxygen and medium components in the mixed fermentation medium were analyzed. Mix cultivation in monosodium glutamate wastewater was further studied. Result indicated 1,600 mg/L of biomass was obtained and 73% of COD were removed.     

Stereoselective production of (S)-1-aralkyl- and 1-arylethanols by freshly harvested and lyophilized yeast cells

Substituted (S)-1-phenyl- 2a–h and (S)-1-benzyl-propan-2-ols 4a and b, and (S)-1-phenylethanol 6 were produced from prochiral ketones 1a–h, 3a,b and 5 by reductions with freshly harvested Zygosaccharomyces rouxii and Debaryomyces hansenii cells. The bioreductions were also performed by lyophilized cells. Comparison of the secondary alcohols from the bioreductions 2b–e,g,h and 4a and authentic (S)-alcohols (S)-2b–e,g,h and (S)-4a synthesized from enantiopure (S)-methyloxirane 7 proved the absolute configuration of the products.     

Continuous production of succinic acid by a fumarate-reducing bacterium immobilized in a hollow-fiber bioreactor

Enterococcus faecalis RKY1, a fumarate-reducing bacterium, was immobilized in an asymmetric hollow-fiber bioreactor (HFBR) for the continuous production of succinic acid. The cells were inoculated into the shell side of the HFBR, which was operated in transverse mode. Since the pH values in the HFBR declined during continuous operation to about 5.7, it was necessary to change the feed pH from 7.0 to 8.0 after 24 h of operation in order to enhance production of succinic acid. During continuous operation with a medium containing fumarate and glycerol, the productivity of succinate was 3.0–10.9 g/(L·h) with an initial concentration of 30 g/L of fumarate, 4.9–14.9 g/(L·h) with 50 g/L of fumarate, and 7.2–17.1 g/(L·h) with 80 g/L of fumarate for dilution rates between 0.1 and 0.4 h⁻¹. The maximum productivity of succinate obtained by the HFBR (17.1 g of succinate /[L·h]) was 1.7 times higher than that of the batch bioconversions (9.9 g of succinate /[L·h]) with 80 g/L of fumarate. Furthermore, the long-term stability of the HFBR was demonstrated with a continuously efficient production of succinate for more than 15 d (360 h).     

Continuous production of butanol by clostridium acetobutylicum immobilized in a fibrous bed bioreactor

We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum. A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9h⁻¹ and pH 4.3. Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.     

Biopreparation of (-)-(1S,3R,4S,6S)-6-hydroxymenthol and (-)-(1S,3R,4S)-1-hydroxymenthol from 1-menthol by Rhizoctonia solani AG-1-IA and IB

Microbial transformation of 1-menthol (1) by six isolates of soil-borne plant pathogenic fungi Rhizoctonia solani AG-1-IA (Rs24, Joichi-2 and RRG97-1) and AG-1-IB (TR22, R147 and 110.4) as a biocatalyst was investigated. Twenty one days precultivation of Rhizoctonia solani AG-1-IA Rs24 and AG-1-IB 110.4 showed excellent yield (98.5-98.6%) of (-)-(1S,3R,4S,6S)-6-hydroxymenthol (2) and (-)-(1S,3R,4S)-1-hydroxymenthol (3) from 1.     

Synthesis and crystal structure of (S)-(+)-2-[(1,1-dimethylethoxy)carbonyl] amino-2-phenylethanol

The preparation and the crystal structure of the title compound are described. C₆H₅CH[NHCOOC(CH₃)₃]CH₂OH is monoclinic with the following unit-cell dimensions: a = 10.354(2), b = 6.533(3), c = 10.505(1) Å, β = 98.58(1)∘. The space group is P2₁ and Z = 2. The crystal-structure determination was run by using 1481 independent reflexions with a final R value of 0.033 (R_w = 0.043).     

Preparation of enantiomerically pure (R)- and (S)-3-amino-3-phenyl-1-propanol via resolution with immobilized penicillin G acylase

Ethyl 2,4-dioxo-4-phenylbutyrate, obtained by condensation of acetophenone with diethyl oxalate, was converted to 3-oxo-3-phenyl-1-propanol in 90% yield by reaction with baker’s yeast. Reductive amination with sodium cyanoborohydride in the presence of ammonium acetate gave the racemic 3-amino-3-phenyl-1-propanol in 65% yield. Enzymatic resolution of the corresponding N-phenylacetyl derivative with penicillin G acylase, immobilized on an epoxy resin gave (S)-amide and (R)-amino alcohol in high enantiomeric purity (ee >99%) and >45% yields for each enantiomer.     

Continuous production of acetaldehyde by immobilized yeast within situ product trapping

Applied Biochemistry and Biotechnology - Two alternative enzymatic routes to continuous biological acetaldehyde production from ethanol by immobilized yeast were compared: ADH ofCandida utilis and...     

Conformational analysis of the NMDA receptor antagonist (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) designed by a novel conformational restriction method based on the structural feature of cyclopropane ring

(1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (2b, PPDC), a new class of potent N-methyl-D-aspartic acid (NMDA) receptor antagonist, was designed based on a new method for restricting the conformation of compounds having a cyclopropane ring. The three-dimensional structures of PPDC obtained by the three different methods of X-ray crystallographic analysis, usual MM2-calculations in vacuum, and MM2 calculations based on the nuclear Overhauser effect (NOE) data in D2O are similar, which are in accord with that hypothesized. These results suggest that this conformational restriction method is particularly effective in designing novel biologically active molecules.     

Biotransformation of (1S)-2-carene and (1S)-3-carene by Picea abies suspension culture

Biotransformation of (1S)-2-carene and (1S)-3-carene by Picea abies suspension culture led to the formation of oxygenated products. (1S)-2-Carene was transformed slowly and the final product was identified as (1S)-2-caren-4-one. On the other hand, the transformation of (1S)-3-carene was rapid and finally led to the formation of (1S)-3-caren-5-one and (1S)-2-caren-4-one as equally abundant major products. The time-course of the reaction indicates that some products abundant at the beginning of the reaction (e.g. (1S, 3S, 4R)-3,4-epoxycarane and (1R)-p-mentha-1(7),2-dien-8-ol) were consumed by a subsequent transformations. Thus, a precise selection of the biotransformation time may be used for a production of specific compounds.     

Ultra-sensitive detection of uranyl ions with a specially designed high-efficiency SERS-based microfluidic device

The uranyl ion(UO_2~(2+)) poses high risks to human health and the environment, hence its detection and monitoring is of utmost significance. However, the development of an ultra-sensitive, high-efficiency and convenient approach for on-site detection of UO_2~(2+) remains a challenge. Herein, a reliable and reusable surface-enhanced Raman spectroscopy(SERS)-based microfluidic biosensor was developed for rapid detection of UO_2~(2+) in real samples. The detection protocol involved the reaction of 5′-Rhodamine B(RhB)-labeled double-stranded DNA for UO_2~(2+)-specific DNAzyme-cleavage reaction in a U-shaped microchannel. Then, the reaction products were delivered into three parallel samples for high-throughput tests by SERS biochips,where 3 D ZnO-Ag mesoporous nanosheet arrays(MNSs) were modified with a single-stranded DNA(ssDNA). The ssDNAwas sequence-complementary with the 5′-RhB-labeled cleaved-stranded DNA(csDNA) from the reaction products. By the hybridization of ssDNA and csDNA, the signal probe RhB was fixed close to the surface of the ZnO-Ag MNSs to enhance the Raman signal. The limit of detection for UO_2~(2+) with the microfluidic-SERS biosensor was 3.71×10~(-15) M. An over 20,000-fold selectivity towards UO_2~(2+) response was also achieved in the presence of 15 other metal ions. The high-throughput microfluidicSERS biosensor operated well for practical UO_2~(2+) detection, with excellent recoveries in contaminated river and tap water from95.2% to 106.3%(relative standard deviation(RSD) 6.0%, n=6). Although the SERS-based microfluidic biosensor developed in this study was deployed for the detection of UO_2~(2+), the reusable and high-efficiency system may be expanded to the detection of other analytes on-site.     

Acylation of (<Emphasis Type="Italic">R,S</Emphasis>)-1-phenylethanol with ethyl acetate over an immobilized enzyme

Acylation of (R,S)-1-phenylethanol, which is a product of acetophenone hydrogenation, over a Pd-supported catalyst, was studied in ethyl acetate with an immobilized lipase. It was demonstrated that in the presence of hydrogen and Pd/C in the reaction medium the (R,S)-1-phenylethanol acylation rate is not hindered, whereas the selectivity was strongly altered in the latter case, leading to significant amounts of side products.