固定化D-氨基酸氧化酶催化DL-氨基酸去消旋化制备非天然L-氨基酸

在过氧化氢酶和氧气存在下,固定化D-氨基酸氧化酶(D-AAO)对映选择性催化DL-氨基酸中的D-对映体氧化脱氨为相应酮酸,L-对映体保留.研究了D-AAO的底物特异性并对反应条件进行了优化.结果表明:D-AAO具有较宽的底物谱,能够催化疏水性α-氨基酸的D-对映体氧化脱氨.在最优反应条件下,D-AAO催化DL-2-氨基丁酸、DL-2-氨基戊酸去消旋化,L-2-氨基丁酸、L-2-氨基戊酸的收率分别为48%和47%,ee分别为99.5%和99.8%.进一步地利用Pd-C/HCOONH4催化氧化脱氨过程中产生的亚氨基酸原位还原,有效提高了L-2-氨基丁酸、L-2-氨基戊酸的收率并保持高的光学纯度.     

Preparation of Non-natural L Amino Acids via Deracemization of DL-Amino Acids Catalyzed by Immobilized D-amino Acid Oxidase

在过氧化氢酶和氧气存在下,固定化D-氨基酸氧化酶（D-AAO）对映选择性催化DL-氨基酸中的D-对映体氧化脱氨为相应酮酸,L-对映体保留.研究了D-AAO的底物特异性并对反应条件进行了优化.结果表明：D-AAO具有较宽的底物谱,能够催化疏水性α-氨基酸的D-对映体氧化脱氨.在最优反应条件下,D-AAO催化DL-2-氨基丁酸、DL-2-氨基戊酸去消旋化,L-2-氨基丁酸、L-2-氨基戊酸的收率分别为48%和47%,ee分别为99.5%和99.8%.进一步地利用Pd-C/HCOONH4催化氧化脱氨过程中产生的亚氨基酸原位还原,有效提高了L-2-氨基丁酸、L-2-氨基戊酸的收率并保持高的光学纯度.     

L-酪氨酸甲酯消旋及Alcalase 2.4L催化对映选择性水解制备D-酪氨酸

提出了一个5-硝基水杨醛催化L-酪氨酸甲酯消旋化的新方法并推测了L-酪氨酸甲酯的消旋机理.在乙腈/磷酸盐缓冲液(pH 7.5)中,5-硝基水杨醛催化L-酪氨酸甲酯消旋为DL-酪氨酸甲酯,消旋率100%,消旋收率93%.优化了Alcalase 2.4L催化DL-酪氨酸甲酯对映选择性水解的反应条件.30℃下,在叔丁醇/磷酸盐缓冲液(pH 7.5)中,Alcalase 2.4L催化DL-酪氨酸甲酯对映选择性水解为L-酪氨酸和D-酪氨酸甲酯.在酶催化水解过程中,L-酪氨酸形成沉淀,容易通过简单的过滤进行分离.D-酪氨酸甲酯在碱性条件下水解为D-酪氨酸,收率91%,ee97%.     

单分散树脂键合手性配体交换色谱固定相拆分DL-氨基酸

以单分散交联聚甲基丙烯酸环氧丙酯-甲基丙烯酸乙二醇双酯(PGMA/EDMA)树脂为基质合成了L-脯氨酸键合手性配体交换固定相,并用于DL-氨基酸的直接光学拆分,考察了流动相pH值、金属离子浓度、流速及温度等因素对DL-氨基酸对映体拆分的影响。结果表明,该固定相在配体交换色谱模式下可对多对DL-氨基酸进行良好的拆分。     

以铜(Ⅱ)-L-羟基脯氨酸配合物为手性选择剂的配体交换毛细管区带电泳拆分α-羟基酸

在使用10mmol/L NH4Ac(pH 4.5),30mmol/L Cu(Ac)2和60mmol/LL-羟基脯氨酸的优化条件下,13min内拆分了3种α-羟基酸对映体(DL-苹果酸、DL-苦杏仁酸和DL-对溴苦杏仁酸),除苹果酸外均获得了满意的拆分结果;考察了电泳缓冲液组成、pH值等影响分离效果的因素。     

手性CdTe量子点制备及在药物青霉胺对映体检测中的应用

以青霉胺对映体作为稳定剂水相制备D-型和L-型CdTe量子点。研究表明,D-型和L-型CdTe量子点的圆二色谱图呈现镜像分布,证明两种量子点是互为光学异构的。D-型量子点(或L-型量子点)与L-型青霉胺(或D-型青霉胺)相互作用将导致其荧光强度的明显下降,而加入同型青霉胺时体系的荧光强度几乎不改变。当D-青霉胺浓度在0.01~0.5 mmol/L之间(或L-青霉胺浓度在0.01~0.2 mmol/L之间)量子点荧光强度峰值与对映体浓度符合线性关系,检出限为0.0033 mmol/L(S/N=3)。方法已成功应用于药物中青霉胺对映体的快速检测。     

正辛基-L-羟基脯氨酸萃取苯丙氨酸的机理及性能

研究了正辛基-L-羟基脯氨酸萃取苯丙氨酸的性能和机理, 详细考察了起始氨基酸的浓度、萃取剂的浓度、酸度、铜离子的浓度、温度等因素分别对D-和L-苯丙氨酸萃取性能的影响. 随着起始氨基酸pH值的增大, 萃取分配比D也增大, 对映体分离系数α可以达到2. 萃取反应为吸热反应, 升高温度有利于反应的进行. 表征了萃合物的组成, 推测萃合物的结构为1∶1∶1型的三元配合物. 研究结果为苯丙氨酸的萃取拆分提供了理论依据.     

解木糖赖氨酸芽孢杆菌发酵和生物催化产生L-2-氨基己二酸

以解木糖赖氨酸芽孢杆菌XX-2为出发菌株，110mmol/L L-赖氨酸单盐酸盐为发酵前体，144h发酵后L-2-氨基己二酸浓度达到10.4mmol/L，产率9.5%。以解木糖赖氨酸芽孢杆菌XX-2全细胞为生物催化剂，利用共生的L-赖氨酸 6-脱氢酶和?-1-哌啶啉-6-羧酸脱氢酶催化L-赖氨酸单盐酸盐转化为L-2-氨基己二酸。最优条件为：细胞浓度45g(干重)/L，L-赖氨酸单盐酸盐浓度100mmol/L，pH7.0，温度30℃，反应时间144h。在最优条件下，从100mmol/LL-赖氨酸单盐酸盐产生90mmol/L L-2-氨基己二酸，产率90%。推测了生物催化过程中L-2-氨基己二酸产生的反应机理。     

高效液相色谱手性固定相法拆分和测定乳酸左氧氟沙星中乳酸对映体

建立了高效液相色谱手性固定相法拆分和测定乳酸左氧氟沙星中乳酸对映体的方法。考察了CuSO₄溶液浓度和异丙醇比例对乳酸对映体和左氧氟沙星分离情况的影响。采用Phenomenex 3126(D)-Penicillamine手性色谱柱，优化流动相为2.5 mmol/L CuSO₄溶液-异丙醇(体积比为93∶7),乳酸对映体达到基线分离。以L-乳酸锂和水解后的D-乳酸为标准品，解决了直接采用乳酸为标准品导致测定结果有偏差的问题。L-乳酸和D-乳酸在20～400μg/mL范围内线性良好，相关系数R²分别为09999和0.9998。重复性实验得到的相对标准偏差(RSD)为0.7%～0.8%,回收率为98.77%～100.1%(RSD≤1.0%),准确度和精密度良好。该方法简便，适用于乳酸左氧氟沙星中乳酸对映体含量的测定。     

改性的配体交换型手性固定相的合成及应用

通过在巯丙基硅胶表面引入苯乙烯长链,再加入甲基丙烯酸缩水甘油酯在聚苯乙烯链端进行延长,用L-脯氨酸和L-羟脯氨酸进行修饰的方法合成了两种聚合物型配体交换型手性固定相(CSPⅣ和CSPⅤ),并采用6种氨基酸对映体考察了合成固定相的色谱拆分效果。结果表明,除DL-脯氨酸外,其余氨基酸对映体均具有相同的出峰顺序,即D-异构体优先出峰。与不含聚苯乙烯单元的配体交换型手性固定相相比,CSPⅣ和CSPⅤ具有更小的保留因子,更好的对映体选择性和更高的分离度。     

Development of nitrilase-mediated process for phenylacetic acid production from phenylacetonitrile

This study aimed at developing an efficient biotransformation process for phenylacetic acid production from phenylacetonitrile by using recombinant Escherichia coli M15 harboring a double mutant MG nitrilase (I113M/Y199G) from Burkholderia cenocepacia J2315. A yield of 2310 U/mL nitrilase was obtained by fermentation after the optimization of cultivation conditions, with a specific activity of 64 U/mg dcw. The MG nitrilase showed high substrate tolerance and completely hydrolyzed 100 mM phenylacetonitrile in 30 min under optimal conditions. To alleviate substrate inhibition, periodic or continuous batch-feeding of substrate was used during the biotransformation. Up to 164 g/L substrate was completely hydrolyzed in 9 h with continuous batch-feeding using resting cells, corresponding to 400 U/mL of nitrilase activity, and leading to production of 163.4 g/L phenylacetic acid. The hydrolysis process has potential application for phenylacetic acid production on a large scale.     

A preliminary information about continuous fermentation using cell recycling for improving microbial xylitol production rates

Xylitolis a sugar-alcohol with important technological properties, such as anticariogenicity, low caloric value, and negative dissolution heat. It can be used successfully in food for mulations and pharmaceutical industries. Its production is therefore in great demand. Biotechnological xylitol production has several economic advantages in comparison with the conventional process based on the chemical reduction of xylose. The efficiency and the productivity of this fermentation chiefly depends on the microorganism and the process conditions employed. In this article a simple continuous culture with cell recycling was evaluated to enhance the capability of Candida guilliermondii FTI 20037 to produce xylitol. The fermentation was initiated batchwise by directly inoculating the grown seed culturein a 2-L bench-scale fermentor. Continuous feeding was begun at a dilution rate (D) of 0.060/h after the xylose concentration had completely consumed and the cell concentration was a bout 4.0 g/L. At a dilution rate of 0.060/h the xylitol concentration was about 15g/L and in creased by about 35%, whereas the dilution rate decreased by about 58%. Furthermore, the volumetric productivity, Q_p, markedly depended on the dilution rate, diminishing by about 37% as D was changed from 0.060 to 0.025/h. These preliminary results show us that the continous fermentation with cell recycling is a good way to study the xylitol production by xylose-fermenting yeasts.     

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.     

Optimization of Saccharification and Fermentation Process in Bioethanol Production from Oil Palm Fronds

Oil Palm Frond (OPF) is one of lignocellulosic biomass, which can be utilized as raw material for bioethanol production. Bioethanol is produced as alternative energy to substitute gasoline. There are four steps in bioethanol production from OPF, i.e pretreatement, saccharification, fermentation and purification process. In this study, optimization of saccharification and fermentation process for OPF was investigated. Two methods and the variations of enzyme concentration were carried out in the saccharification and fermentation process. Separate hydrolysis and fermentation process (SHF) and simultaneous saccharification and fermentation process (SSF) were conducted to produce ethanol optimally. Variations of enzyme concentration used in this process were 10, 20, 30 and 40 FPU/g substrate. The result shows that the highest ethanol concentration can be obtained in SSF process with 30 FPU/g substrate of enzyme concentration. The process produced 59.20 g/L ethanol (95.95% yield ethanol) at 96 h of SSF process.     

Biotechnological Production of 20-alpha-Dihydrodydrogesterone at Pilot Scale

The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α-dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70 L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190 μM day⁻¹ was determined for a total biotransformation time of 136 h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3 ± 1.4 g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale.     

Enhancement of 1,3-Dihydroxyacetone Production by a UV-induced Mutant of Gluconobacter oxydans with DO Control Strategy

The 1,3-dihydroxyacetone (DHA)-overproducing mutant of Gluconobacter oxydans was screened via UV mutagenesis to enhance the DHA production, and the DHA fermentation condition was optimized using the dissolved oxygen (DO) control strategy. The stable mutant G. oxydans ZJB11001 exhibits high DHA productivity and can tolerate high DHA concentrations. The optimal condition for DHA production by G. oxydans ZJB11001 in a 15-L fermentor required an initial medium containing 5 g/L yeast extract, 20 g/L glycerol, 0.5 g/L K(2)HPO(4), 0.1 g/L MgSO(4)·7H(2)O. The glycerol feeding rate was manually controlled to maintain the glycerol concentration at 5-10 g/L range. The culture pH was maintained at 6.0 within the first 20 h, and then adjusted to 5.0 until the end of the fermentation. The DO concentration increased from 20% to 30% after 24 h of fermentation, and then to 40% after 60 h of fermentation. The maximum DHA concentration of 209.6 ± 6.8 g/L was achieved after 72 h of fed-batch fermentation at 30 °C.     

Butanol Production from Cane Molasses by <Emphasis Type="Italic">Clostridium saccharobutylicum</Emphasis> DSM 13864: Batch and Semicontinuous Fermentation

Clostridium acetobutylicum strains used in most Chinese ABE (acetone–butanol–ethanol) plants favorably ferment starchy materials like corn, cassava, etc., rather than sugar materials. This is one major problem of ABE industry in China and significantly limits the exploitation of cheap waste sugar materials. In this work, cane molasses were utilized as substrate in ABE production by Clostridium saccharobutylicum DSM 13864. Under optimum conditions, total solvent of 19.80 g/L (13.40 g/L butanol) was reached after 72 h of fermentation in an Erlenmeyer flask. In a 5-L bioreactor, total solvent of 17.88 g/L was attained after 36 h of fermentation, and the productivity and yield were 0.50 g/L/h and 0.33 g ABE/g sugar consumption, respectively. To further enhance the productivity, a two-stage semicontinuous fermentation process was steadily operated for over 8 days (205 h, 26 cycles) with average productivity (stage II) of 1.05 g/L/h and cell concentration (stage I) of 7.43 OD₆₆₀, respectively. The average batch fermentation time (stage I and II) was reduced to 21−25 h with average solvent of 15.27 g/L. This study provides valuable process data for the development of industrial ABE fermentation process using cane molasses as substrate.     

Biotechnological production of xylitol from agroindustrial residues

Batch, fed-batch, and semicontinuous fermentation processes were used for the production of xylitol from sugarcane bagasse hemicellulosic hydrolysate. The best results were achieved by the semicontinuous fermentation process: a xylitol yield of 0.79 g/g with an efficiency of 86% and a volumetric productivity of 0.66 g/L/h.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

Evaluation of cell recycle on Thermomyces lanuginosus xylanase a production by Pichia pastoris GS 115

This work aims to evaluate cell recycle of a recombinant strain of Pichia pastoris GS115 on the Xylanase A (XynA) production of Thermomyces lanuginosus IOC-4145 in submerged fermentation. Fed-batch processes were carried out with methanol feeding at each 12h and recycling cell at 24, 48, and 72 h. Additionally, the influence of the initial cell concentration was investigated. XynA production was not decreased with the recycling time, during four cell recycles, using an initial cell concentration of 2.5 g/L. The maximum activity was 14,050 U/L obtained in 24h of expression. However, when the initial cell concentration of 0.25 g/L was investigated, the enzymatic activity was reduced by 30 and 75% after the third and fourth cycles, respectively. Finally, it could be concluded that the initial cell concentration influenced the process performance and the interval of cell recycle affected enzymatic production.