Production of L-erythrose via L-erythrulose from erythritol using microbial and enzymatic reactions

A rare aldotetrose, L-erythrose, was produced from erythritol via a two-step reaction. In the first step, complete oxidation of erythritol to L-erythrulose was achieved by using Gluconobacter frateurii IFO 3254. Washed cell suspension of the strain grown on tryptic soy broth (TSB) supplemented with 1% d-sorbitol was used to carry out the transformation reaction at 30 degrees C with shaking at 170 rpm. At 10% substrate concentration, 98% erythritol was converted to L-erythrulose within 48 h. The produced L-erythrulose was then used as a substrate for the production of L-erythrose. The isomerization of L-erythrulose to L-erythrose was carried out using constitutively produced L-ribose isomerase (l-RI) from the mutant strain Acinetobacter sp. DL-28 grown on D-lyxose mineral salt medium. At equilibrium, the yield of L-erythrose from L-erythrulose was 18% and finally 1.7 g L-erythrose was obtained from 10 g erythritol. After a number of simple purification steps, the product was isolated from the reaction mixture by ion-exchange column chromatography (Dowex 50W-X2, Ca2+). The structure of the product was determined after NaBH4 reduction from Infrared (IR) and 13C nuclear magnetic resonance (NMR) spectra.     

High-level production of erythritol by strain 618A-01 isolated from pollen

We have isolated a microorganism (strain 618A-01) from pollen which has the ability to produce erythritol when grown in the presence of glucose as the carbon source. When cultivated in a medium consisting of 20% glucose and 1% dried bouillon in a shake flask, 75 g/l erythritol was produced after 950 h, corresponding to a 37.5% yield against glucose consumption. No other polyols, including glycerol, were detected in the medium. Positive-ion fast atom bombardment mass spectrometry and 1H- and 13C-NMR analyses confirmed that the fermentation product was erythritol. Scanning electron microscopic analysis clearly demonstrated that the cells grown on YPD medium at 30 degrees C showed yeast-like morphology, while they appeared like hyphae at 37 degrees C. The complete 18S rRNA sequence of the isolate was determined, which showed high identity (99.5%) with the genus Ustilago of the phylum Basidiomycota. The data strongly suggest that strain 618A-01 belongs to the class Ustilaginomycetes. The culture conditions for the production of erythritol by the isolate were examined. The use of medium containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl yielded the highest cell growth and erythritol productivity among the media tested. Continuous glucose feeding at 6-7% to the fermentor further increased the production of erythritol, and we obtained a maximal 100 g/l erythritol after 530 h, with a 39.3% yield.     

耐高渗酵母产赤藓糖醇的影响因素

球拟酵母OS-194是一株单产赤藓糖醇的耐高渗酵母,该菌株高产赤藓糖醇的最佳培养基配方为葡萄糖10g/dL,酵母膏0.5g/dL,尿素0.1g/dL.最适培养条件是在摇瓶转速150r/min的条件下于35℃培养4d.在上述培养条件下,该菌株赤藓糖醇的耗糖转化率高达29.6%.磷是限制OS-194菌株高产赤藓糖醇的主要因素,当培养液中的磷质量浓度低于31.5mg/L时,赤藓糖醇的产量最高;随着磷质量浓度的升高,该菌株赤藓糖醇的产量降低,而酒精的产量和生物量却有明显升高.同时,OS-194菌株还能利用果糖、蔗糖和D-甘露糖产赤藓糖醇.     

Production of L-sorbitol from L-fructose by Aureobasidium pullulans LP23 isolated from soy sauce mash

A strain LP23 that can convert L-fructose to L-sorbitol was isolated from soy sauce mash and identified as Aureobasidium pullulans. The cells grown on L-arabinose were found to have relatively high L-fructose to L-sorbitol conversion potential. Addition of erythritol to the reaction mixture considerably accelerated the conversion rate of L-fructose to L-sorbitol. During the conversion reaction, erythritol was added to the reaction mixture at 8-h intervals to maintain the concentration of erythritol at 1.0%. The final conversion ratios were 82.8%, 95.3%, 92.4%, and 42.6% using washed cells when the concentrations of L-fructose were 1.0%, 2.0%, 5.0% and 10.0%, respectively. The product from L-fructose was identified as L-sorbitol by HPLC analysis, infrared spectroscopy, optical rotation and melting point measurements.     

高产赤藓糖醇菌株RH-UV-L4-F9发酵条件的优化

以高产赤藓糖醇菌株RH-UV-L4-F9为研究对象,采用生物统计方法分别对该菌株的发酵培养基和培养条件进行优化。发酵培养基最佳配比为葡萄糖30%,酵母膏0.5%,脲1%,MgSO_4 0.05%;最适发酵条件为34℃,初始pH值6.0.摇床转数180r/min。最适条件下赤藓糖醇产量为157.4mg/mL。     

白酒双边固态酒精发酵动力学模型的构建

旨在提出一种数学建模方法,以描述白酒双边固态酒精发酵过程。该研究基于实验室模拟发酵数据,创新性引入酶动力学方程,建立白酒双边固态酒精发酵的动力学模型,描述酵母生长、葡萄糖转化、产物乙醇形成和底物淀粉水解的变化过程,并采用1stOpt的两种不同算法对模型参数进行辨识和拟合。结果表明,引入酶动力学模型使得拟合结果得到了很大的提升。通用全局算法(UGO)的拟合结果优于遗传算法(GA),且参数辨识结果更具有实际意义。采用UGO得到淀粉水解、葡萄糖转化、酵母生长、乙醇生产4条拟合曲线的拟合优度R²分别达到0.986 52、0.992 65、0.910 52和0.996 45,表明该动力学模型能较好的定量描述白酒双边固态酒精发酵过程,对其过程的优化与控制具有一定的实际意义。     

赤藓糖醇对柠檬汁饮料中维生素C保护作用的研究

本文以柠檬汁饮料作为实验材料,采用调节温度的方法对维生素C的热降解进行动力学研究。在建立动力学模型的基础上,通过速率常数、反应活化能等动力学参数,来探究柠檬汁饮料中赤藓糖醇的加入对维生素C的保护作用。研究结果表明:柠檬汁饮料在贮藏过程中,维生素C对热不稳定,其热力学降解符合一级反应动力学模型;在本次实验中,1%~3%赤藓糖醇浓度的加入均有助于减缓维生素C的降解速率。其中,当赤藓糖醇添加浓度为2%时,维生素C降解反应活化能达到最大,为75.47kJ/mol,比对照高出了9.79kJ/mol。因此,赤藓糖醇在一定程度上能够起到对维生素C的保护作用。      

Generation of an antibody specific to erythritol, a non-immunogenic food additive

Erythritol, a simple sugar alcohol, is widely used as a food and drug additive owing to its chemical inertness, sweetness and non-toxicity. Adverse reactions to erythritol are rare and only three cases of allergic reactions to foods containing erythritol have been reported. Being inert, erythritol cannot produce an immunological response. In order to explain the mechanism of immunogenicity of erythritol, a method to obtain erythritol epitopes on a carrier protein, which can serve as an immunogen to develop antibodies against erythritol, is described. D-Erythrose was conjugated to bovine serum albumin at pH 8 by reductive amination. The reduction product of the Schiff base of D-erythrose-bovine serum albumin conjugate creates erythritoyl groups. Rabbits immunized with erythritol-bovine serum albumin conjugate (29 haptens/molecule) showed good antibody response (detection of 1 µg antigen, erythritol-keyhole limpet haemocyanin conjugate possessing 50% modified amino groups, at 1 : 50 000 dilution). Anti-erythritol immunoglobulin-G antibodies were purified from the immune serum using hapten-affinity chromatography on an erythritol-keyhole limpet haemocyanin-Sepharose CL-6B affinity matrix. The yield of erythritol-specific antibody was approximately 40 µg ml^-1 of rabbit antiserum. Enzyme-linked immunobsorbant assay inhibition studies using sugars, sugar alcohols and L-lysine showed minimal cross-reactivity (approximately 4%) when compared with erythritol; only dithioerythritol showed a cross-reactivity of approximately 33%. D-Threitol and L-threitol (isomers of erythritol) had cross-reactivities of 15 and 11%, respectively. The inhibition studies confirmed the haptenic nature of erythritol and indicated that the erythritoyl group is a single epitope. The reaction scheme outlined here for the generation of erythritol epitopes appears to provide a basis for the immunogenicity of erythritol.     

麦芽根提取5'-磷酸二酯酶对RNA酶解工艺研究

从麦芽根提取中的5′-磷酸二酯酶,原酶液酶活达到300U/mL。将提取的酶液用于水解酵母RNA,得到5′-AMP、UMP、CMP、GMP四种核苷酸,并系统研究了麦芽根5′-磷酸二酯酶对核酸的酶解条件。结果表明,最佳反应条件为:底物浓度3%,酶用量5%,温度68℃,pH6.5,水解时间为4～5h,在此条件下,酶解率可以达到84%。在酶解2h内补加RNA底物可提高酶利用率。     

赤藓糖醇高产菌选育及发酵条件研究

为了提高赤藓糖醇的产量,对自选耐高渗酵母菌T-3-2进行紫外线与亚硝酸复合诱变处理,并采用响应曲面法优化了其发酵条件。复合诱变得到1株稳定高产突变株UN-11,其赤藓糖醇的产量达到78.3mg/mL,比T-3-2提高了38.8%;通过响应面分析建立了关键影响赤藓糖醇产量的二次多项式数学模型,得到最佳生产工艺条件为:发酵温度31℃、转速170r/min、接种量9.8%。模型预测结果产物浓度达到84.93mg/mL,验证实验结果产物浓度为85.25mg/mL。该模型对赤藓糖醇工业化生产有一定的指导意义。      

Emulsion culture: a miniaturized library screening system based on micro-droplets in an emulsified medium

A typical library screen in directed evolution primarily requires physical separation of the clones on agar plates followed by detection of clones with improved properties; using this method only limited numbers of clones relative to the number of potential variations can be assessed. In particular, screening for a secretory enzyme is difficult to perform at high clone density, because of diffusion of the signal or unfavorable utilization of the reaction product by neighboring clones. In this study, we have developed a novel method of enrichment culture: “Emulsion Culture”, i.e., segregated replication of clones in an emulsified culture medium. Clones expressing enzyme-variants are separately distributed to small (up to 50 μm in diameter), segregated compartments composed of a droplet of medium to form several tens of millions of microcolonies in a milliliter of medium, which allows a miniaturized, in-bulk screening of clones. We applied this culture method to yeast clones expressing secretory beta-galactosidase to analyze the enrichment factor achieved. A high-density screen for a signal peptide sequence that maximizes extracellular production of the enzyme was also performed to demonstrate the practicability of this culture method. In addition, micro-channel emulsification was tested as a method of forming uniformly-sized compartments in the emulsion.     

冷冻保护剂和储藏条件对海洋酵母冻干粉活性的影响

海洋拮抗酵母Rhodosporidium paludigenum作为生防保鲜剂在控制水果采后真菌类病原菌中表现出了良好的抑制效果,然而生防制剂要真正取代化学杀菌剂,必须以产品的形式投入市场,即实现其商品化生产。实验将该拮抗酵母制成冻干粉,研究冻干保护剂和储藏条件对海洋酵母干粉稳定性的影响。结果显示,海藻糖作为保护剂可以显著地提高酵母干粉的存活率和生防活性,用5%的海藻糖作保护剂,存活率为42%,而对照仅为3%。研究还发现,酵母细胞的存活率与其胞内海藻糖含量具有一定的相关性。另外,储藏实验表明,低温(4℃)可以较好地保持酵母干粉的存活力。而常温(25℃)下储藏1个月,每克干粉中酵母活细胞数下降5个数量级,因此常温不适合储藏该新型拮抗酵母生防制剂。     

白首乌酵素发酵工艺的优化

为了开发一款白首乌酵素产品,本文以白首乌为原料,采用乳酸菌和酵母菌复合发酵制备酵素,对乳酸菌和酵母菌的单因素实验条件进行优化,并确定两菌种复合发酵时的接种顺序和发酵时间。结果表明:采用先接种酵母菌后接种乳酸菌发酵的方式最优,该接种顺序下的最佳复合发酵工艺条件为:梅山酵母菌(S4)在初始pH5.0、装瓶量30 mL(250 mL)、蛋白胨添加量1.2%、接种量0.2%、发酵温度30 ℃的条件下,发酵24 h,酵母菌发酵结束后,接种3%的复合乳酸菌(嗜酸乳杆菌(LA)和植物乳杆菌(LP)以1:1混合(v:v)),37 ℃静置发酵36 h,然后在4~8 ℃下后发酵12 h,使发酵液产香。在此条件下,白首乌酵素中乳酸菌活菌为2.61×10⁸ CFU/mL,酵母菌活菌数为7.9×10⁷ CFU/mL,超氧化物歧化酶(superoxide dismutase,SOD)活力为4.23×10⁴ U/L。采用发酵的方法制备白首乌产品,为白首乌资源的开发利用提供了新的思路,对促进白首乌产业的发展具有重要意义。     

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.     

无溶剂体系酶法催化酸解合成共轭亚油酸甘油酯

采用商业化固定化酶Novozym 435作为生物催化剂,催化共轭亚油酸(CLA)和葵花籽油的酸解反应合成富含CLA的结构脂质(CLA-SL).研究了在无溶剂体系中,底物摩尔比、酶用量、体系含水量、反应温度和反应时间对产物中CLA含量和Sn-2位CLA含量的影响.结果表明,最佳反应条件为:CLA与葵花籽油摩尔比3 :1,酶用量10%,体系含水量1%,反应温度55 ℃,反应时间36 h.在最佳反应条件下,产物中的CLA含量和Sn-2位CLA含量分别为15.7%和2.73%.     

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA-triphosphatase activities.

The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5'-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid-specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements.     

Key role for transketolase activity in erythritol production by Trichosporonoides megachiliensis SN-G42

Erythritol is an important sugar alcohol industrially produced only by fermentation. The highly osmophilic yeast-like fungi, Trichosporonoides megachiliensis SN-G42, enables commercial production of erythritol with a high conversion from glucose to erythritol of more than 47%. However, the microbial production pathway of erythritol remains unclear. In the present study, the activities of enzymes in the pentose phosphate pathway of Trichosporonoides megachiliensis SN-G42 used for industrial erythritol production were measured under various culture conditions to examine the production mechanism and the key-enzymes.As a result, the various enzyme activities of this organism are revealed in the pentose phosphate pathway, i.e., those of hexokinase, glucose-6-phosphate dehydrogenase, gluconate dehydrogenase, transketolase, transaldolase, and erythrose reductase. In the cultures in which erythritol was produced after completion of cell growth, the enzyme activities of the pentose phosphate pathway were higher than those of the TCA cycle. In particular, transketolase activity was correlated with erythritol productivity under various production cultures with different agitation speeds and thiamine concentrations.These results suggest that erythritol may be produced mainly through the pentose phosphate pathway. In addition, the high activity of transketolase is required to produce abundant intermediates, which results in high erythritol productivity. As such, transketolase appears to be a key-enzyme for erythritol production in the organism studied.     

一株西沙群岛野生诺尼种子内生细菌CICC 10707的分离与多相鉴定

对分离自我国西沙群岛野生诺尼种子的内生细菌菌株CICC 10707的分类地位进行了研究,为今后研究该菌的生物学功能奠定了基础。利用基于形态学、分子生物学及生理生化特征的微生物多相分类鉴定技术,将其鉴定为草螺菌属(Herbaspirillum sp.)中的织片草螺菌(Herbaspirillum seropedicae),该菌具有氧化酶和接触酶活性,且能够利用甘露糖、葡萄糖、赤藓糖醇等多种碳源物质。      

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

A rapid method for localized mutagenesis of yeast genes.

We have developed a simple procedure for the localized mutagenesis of yeast genes. In this technique the region of interest is first amplified under mutagenic polymerase chain reaction (PCR) conditions. Cotransformation of the PCR product with a gapped plasmid containing homology to both ends of the PCR product allows in vivo recombination to repair the gap with the mutagenized DNA. This procedure is efficient, allows targeting of specific regions for mutagenesis, and requires no subcloning steps in Escherichia coli.