Microbial removal of ionic mercury in a three-phase fluidized bed reactor

The reductive biotransformation of mercuric ions to elemental mercury was studied by applying a model system with a genetically engineered Pseudomonas putida strain in a lab scale three-phase fluidized bed (TPFB). The aim was to demonstrate the suitability of the TPFB to demercurize effluent streams containing up to 10 mg Hg2+ dm(-3). The TPFB is used, first, to carry out the biotransformation on the alginate immobilized biocatalyst and, second, to remove the produced Hg0 by volatilization into the gas phase followed by its recovery through fast oxidative absorption. Targeted experiments with the immobilized biocatalyst were designed and carried out to determine mercury adsorption data on the biomass and all relevant mass transport rates at conditions prevailing in the TPFB. The evaluation of the performance data in the TPFB revealed almost complete reaction control and hence negligibility of mass transfer resistances. This simplifies the scale-up of larger TPFB reactors for mercury removal as it can be based on the known kinetics alone. The measured biotransformation capacities in the TPFB are similar to those reported for the fixed bed technology which has already proven its applicability at an industrial scale in long time runs. However, the TPFB offers some advantages over the fixed bed and could therefore possibly be a favorable, reliable, and less costly alternative to the existing technology.     

Stability evaluation of an immobilized enzyme system for inulin hydrolysis

The stability of free and Amberlite-immobilized inulinase, aiming at inulin hydrolysis was evaluated. The apparent activation energy of the biotransformation decreased when the immobilized biocatalyst was used, suggesting diffusional limitations, despite a decrease in the optimal temperature for catalytic activity for the immobilized biocatalyst. Thermal deactivation, of both forms of the biocatalyst, was evaluated by the linear inverted model. Inulinase immobilization consistently enhanced half-life of the enzyme, which increased up to 6-fold, as compared to the free form. Mean enzymatic activity was computed for both forms of the biocatalyst, and evidenced a decrease of optimal temperature with increased incubation periods. The deactivation energies estimated by an Arrhenius plot, evidenced a decrease of roughly 20% when free inulinase was used. The immobilized biocatalyst was effectively reused in successive batch runs for the hydrolysis of a 5% inulin solution.     

根皮苷生物转化制备根皮素工艺优化研究

研究酿酒酵母（Saccharomyces cerevisiae）生物转化根皮苷制备根皮素的工艺条件。以根皮素产率为考察指标,采用单因素试验考察转化时间、转化温度、底物质量浓度和液料比,然后采用响应面分析法对根皮素制备工艺条件进行优化。结果表明,根皮苷生物转化最佳工艺条件确定为：转化时间18 h、转化温度41 ℃、液料比12∶1（V/V）,底物质量浓度8 g/L。在此条件下,根皮素产率可达70.12%,与模型理论值（70.97%）基本相符。利用本方法制备根皮素,成本低、转化率高,为工业化的生产提供理论依据。     

Kefir Immobilized on Corn Grains as Biocatalyst for Lactic Acid Fermentation and Sourdough Bread Making

Abstract: The natural mixed culture kefir was immobilized on boiled corn grains to produce an efficient biocatalyst for lactic acid fermentation with direct applications in food production, such as sourdough bread making. The immobilized biocatalyst was initially evaluated for its efficiency for lactic acid production by fermentation of cheese whey at various temperatures. The immobilized cells increased the fermentation rate and enhanced lactic acid production compared to free kefir cells. Maximum lactic acid yield (68.8 g/100 g) and lactic acid productivity (12.6 g/L per day) were obtained during fermentation by immobilized cells at 37 °C. The immobilized biocatalyst was then assessed as culture for sourdough bread making. The produced sourdough breads had satisfactory specific loaf volumes and good sensory characteristics. Specifically, bread made by addition of 60% w/w sourdough containing kefir immobilized on corn was more resistant regarding mould spoilage (appearance during the 11^th day), probably due to higher lactic acid produced (2.86 g/Kg of bread) compared to the control samples. The sourdough breads made with the immobilized biocatalyst had aroma profiles similar to that of the control samples as shown by headspace SPME GC‐MS analysis.     

酿酒酵母表面展示β-淀粉酶及其酶学性质研究

通过PCR技术扩增来源于大麦的β-淀粉酶基因,将其与酿酒酵母细胞壁蛋白α凝集素基因在读框内融合,构建得到表面展示载体pBA-AG,进一步将该重组质粒通过遗传转化,整合到酿酒酵母W303-1A的染色体中,获得了β-淀粉酶经过α凝集素锚定信号结合到细胞壁上的重组酵母。重组酵母表面展示的β-淀粉酶活力为131U/g干细胞。对展示的β-淀粉酶酶学性质研究表明,其最适反应温度为50℃,最适作用pH为5.0,与游离酶相比,其温度稳定性和pH稳定性均得到提高。本研究利用α凝集素系统首次将β-淀粉酶成功展示在酿酒酵母表面,为以酿酒酵母为基础的全细胞催化剂研究与应用打下了一定基础。     

Advances in pullulan production from agro-based wastes by Aureobasidium pullulans and its applications

Pullulan is an important exopolysaccharide commercially obtained from ubiquitous fungi Aureobasidium pullulans. Pullulan has unique physicochemical properties, due to which it has wide applications in the food, pharmaceutical and biomedical industries. Various synthetic media and agro-industrial wastes have been utilized for the production of pullulan. Agro-industrial waste has become a preferred substrate for biotransformation due to its economical aspect and nutritional attributes. Pullulan is one of the valuable products produced by the biotransformation of agro-based wastes. Numerous factors which influence the pullulan production are the type of substrate, the addition of carbon source, nitrogen source, pH of the medium, aeration/ agitation speed and temperature. The cost, productivity of the product and efficiency depends on the selection of raw material, type of fermentation (bioprocess), type of strain and downstream processing (recovery). This review focuses on pullulan production from agro-based wastes, the influence of factors and its applications.     

Glycyrrhetic Acid 3‐O‐Mono‐β‐d‐glucuronide (GAMG): An Innovative High‐Potency Sweetener with Improved Biological Activities

Glycyrrhetic acid 3‐O‐mono‐β‐d ‐glucuronide (GAMG) is an important derivative of glycyrrhizin (GL) and has attracted considerable attention, especially in the food and pharmaceutical industries, due to its natural high sweetness and strong biological activities. The biotransformation process is becoming an efficient route for GAMG production with the advantages of mild reaction conditions, environmentally friendly process, and high production efficiency. Recent studies showed that several β‐glucuronidases (β‐GUS) are key GAMG‐producing enzymes, displaying a high potential to convert GL directly into the more valuable GAMG and providing new insights into the generation of high‐value compounds. This review provides details of the structural properties, health benefits, and potential applications of GAMG. The progress in the development of the biotransformation processes and fermentation strategies to improve the yield of GAMG is also discussed. This work further summarizes recent advances in the enzymatic synthesis of GAMG using β‐GUS with emphasis on the physicochemical and biological properties, molecular modifications, and enzymatic strategies to improve β‐GUS biocatalytic efficiencies. This information contributes to a better framework to explore production and application of bioactive GAMG.     

代谢工程在酿酒酵母茵育种中的应用研究进展

酿酒酵母(Saccharomyces cerevisiae)是传统的乙醇生产重要菌株,也是第1个完成基因组测序的真核生物。随着世界能源储备的日益枯竭,燃料乙醇作为液体可再生能源的广泛应用,利用生物技术对酿酒酵母进行遗传修饰,改变其代谢网络,构建高产乙醇的转基因酿酒酵母已成为当今酿酒酵母分子育种的热点。针对近年来利用代谢工程拓展酿酒酵母底物利用范围、降低副产物生成率、提高乙醇产量、改良细胞特性以及缩短发酵周期等方面的研究进展予以综述。     

Biotransformation of monoterpene alcohols by Saccharomyces cerevisiae, Torulaspora delbrueckii and Kluyveromyces lactis

Monoterpenoids are important flavour compounds produced by many plant species, including grapes (Vitis vinifera) and hops (Humulus lupulus). Biotransformation reactions involving monoterpenoids have been characterized in filamentous fungi, but few examples have been observed in yeasts. As monoterpenoids are in contact with yeasts during beer and wine production, biotransformation reactions may occur during the fermentation of these beverages. This paper describes the biotransformation of monoterpene alcohols, of significance in the alcoholic beverage industries, by three yeast species. All three species analysed had the ability to convert monoterpenoids. Saccharomyces cerevisiae and Kluyveromyces lactis reduced geraniol into citronellol, whilst all three yeasts produced linalool from both geraniol and nerol. Monocyclic alpha-terpineol was formed from both linalool and nerol, by all three yeasts. alpha-Terpineol was then converted into the diol cis-terpin hydrate. K. lactis and Torulaspora delbrueckii also had the ability to form geraniol from nerol. Finally, the stereospecificity of terpenoid formation was analysed. Both (+) and (-) enantiomers of linalool and alpha-terpineol were formed in roughly equal quantities, from either geraniol or nerol.     

Increase in pladienolide D production rate using a Streptomyces strain overexpressing a cytochrome P450 gene

Pladienolide B and its 16-hydroxylated derivative (pladienolide D) are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107 showing strong in vitro and in vivo antitumor activity. While pladienolide B is mainly produced by this strain, pladienolide D is produced to a lesser extent. To facilitate the production of pladienolide D by biotransformation, we found that Streptomyces bungoensis A-1544 was able to hydroxylate pladienolide B at 16-position. We identified psmA from S. bungoensis A-1544, which encoded a pladienolide B 16-hydroxylase PsmA belonging to the CYP105 family of cytochrome P450. To increase the efficiency of pladienolide D production, we constructed recombinant S. bungoensis A-1544 overexpressing psmA and performed biotransformation of pladienolide B to pladienolide D. This biotransformation achieved a production level 15-fold higher than that using the control strain S. bungoensis A-1544/pIJ702.     

粟酒裂殖酵母转化底物茄尼醇合成CoQ_(10)的研究

研究了水/有机反应体系中,以茄尼醇为底物时,粟酒裂殖酵母(Schizosaccharomyces promb)2.1794转化合成CoQ10产量情况,并对其转化工艺进行了初步的探讨。确定了适宜于粟酒裂殖酵母转化茄尼醇生成CoQ10的条件为:异丙醇和pH 5.0、0.1 mol/L的磷酸缓冲液按2∶8的体积比形成转化体系,菌体添加量的质量浓度为0.5%,底物添加质量浓度为500 mg/L,30℃下220 r/min转化8 h,CoQ10产量为91.037 mg/L(发酵液),单位细胞内CoQ10产量为3.726 mg/g(湿细胞),与空白相比提高了203.45%,与在水相转化体系中相比提高了81.24%。     

共培养时酿酒酵母对速生梭菌己酸代谢的影响及其机理

以1 株可以产己酸的速生梭菌（Clostridium celerecrescens）JSJ-1与1 株酿酒酵母（Saccharomyces cerevisiae）C-1为研究对象,在不同营养条件下,比较2 种微生物纯培养、共培养过程中生长代谢（菌落数、葡萄糖、乙醇、丁酸、己酸）的差异,分析S. cerevisiae对己酸菌己酸代谢的影响及其机理。结果发现：在厌氧条件下,34 ℃静置培养,S. cerevisiae C-1比C. celerecrescens JSJ-1更具有生长优势,会优先利用培养基中的葡萄糖。当培养基中唯一碳源为葡萄糖时,S. cerevisiae C-1会利用葡萄糖代谢生成乙醇,为C. celerecrescens JSJ-1合成己酸提供底物。当培养基中含0.5%葡萄糖和2%乙醇时,共培养相比C. celerecrescens JSJ-1单独培养,己酸的生成时间提前了4 d。葡萄糖对C. celerecrescens JSJ-1生成己酸有较强的抑制作用,共培养时S. cerevisiae C-1利用葡萄糖可缓解葡萄糖对己酸生成的抑制。在浓香型白酒发酵过程中,S. cerevisiae不仅可以为己酸菌合成己酸提供底物,而且可以缓解葡萄糖对己酸生成的抑制作用。     

Generation of Flavors and Fragrances Through Biotransformation and De Novo Synthesis

Flavors and fragrances are the result of the presence of volatile and non-volatile compounds, appreciated mostly by the sense of smell once they usually have pleasant odors. They are used in perfumes and perfumed products, as well as for the flavoring of foods and beverages. In fact the ability of the microorganisms to produce flavors and fragrances has been described for a long time, but the relationship between the flavor formation and the microbial growth was only recently established. After that, efforts have been put in the analysis and optimization of food fermentations that led to the investigation of microorganisms and their capacity to produce flavors and fragrances, either by de novo synthesis or biotransformation. In this review, we aim to resume the recent achievements in the production of the most relevant flavors by bioconversion/biotransformation or de novo synthesis, its market value, prominent strains used, and their production rates/maximum concentrations.     

Isolation and nucleotide sequence of a gene encoding tRNA nucleotidyltransferase from Kluyveromyces lactis

A gene (KlCCA1) encoding ATP(CTP):tRNA specific tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated from Kluyveromyces lactis by complementation of the Saccharomyces cerevisiae cca1-1 mutation. Sequencing of a 2665 bp EcoRI-SpeI restriction fragment revealed an open reading frame potentially encoding a protein of 489 amino acids with 57% sequence similarity to its S. cerevisiae homologue. Southern hybridization revealed a single copy of KlCCA1 in the K. lactis genome. KlCCA1 was able to complement both the mitochondrial and cytosolic defects in the cca1-1 mutant, suggesting that, as in S. cerevisiae, the K. lactis gene encodes a sorting isozyme that is targeted to mitochondria and the nucleus and/or cytosol. An altered KlCCA1 gene encoding a tRNA nucleotidyltransferase that lacked its first 35 amino acids was able to complement the nuclear/cytosolic but not the mitochondrial defect in the S. cerevisiae cca1-1 mutant, suggesting that the 35 amino-terminal amino acids are necessary for targeting to mitochondria but are not required for enzyme activity. Our results suggest that the mechanisms for production and distribution of mitochondrial and nuclear/cytosolic tRNA nucleotidyltransferase in K. lactis differ from those seen in S. cerevisiae.     

高产SO2啤酒酵母菌株发酵性能的研究

主要对高产二氧化硫啤酒酵母突变株M8的发酵性能和遗传稳定性进行了研究.啤酒发酵实验结果表明,突变株M8发酵性能较好.突变株M8第20代菌株发酵的啤酒的二氧化硫生成量比原株S-5提高了20.72%,硫化氢生成量降低了49.3%,遗传稳定性良好.     

酿酒酵母醇酰基转移酶的研究进展

酵母发酵产生酯类化合物是白酒发酵过程中的重要产香成分之一。在白酒多年的发展过程中,其香味物质的成分一直不能为人们标准化,不能实现更好的机械化智能化的生产,因此研究酵母产酯途径已成为人们现在热议的话题。该文综述了酿酒酵母酰基酯合成途径中的关键酶的酶学性质、晶体结构与分子催化机制,并展望了其在提升白酒品质和推进白酒的标准化进程的潜力与价值。     

构建高效糖配体再生重组菌生物催化合成莱鲍迪苷D

利用拟南芥（Arabidopsis thaliana）蔗糖合酶AtSUS3构建活化糖配体尿苷二磷酸葡萄糖（uridine diphosphate glucose,UDPG）再生体系,与高效催化莱鲍迪苷D（rebaudioside D,RD）合成的糖基转移酶协同偶联完成RD的高效生物催化。从拟南芥cDNA克隆获得AtSUS3基因,将其装配至pET28a获得表达质粒pLW105,随后pLW105与携带糖基转移酶EUGT11基因的质粒共转化大肠杆菌BL21（DE3）构建双酶共表达工程菌。在不添加UDPG或者尿核苷二磷酸（uridine diphosphate,UDP）的条件下,以上述双酶共表达工程菌全细胞催化莱鲍迪苷A（RA）获得的底物摩尔转化率大于80%,RD产量达到930?mg/L。随后构建双酶基因串连质粒pLW108及双酶共表达大肠杆菌BL21（DE3）工程菌。用串连质粒构建的工程菌催化效果与双质粒共转化相同。采用共表达双酶工程菌的发酵破碎粗酶进行催化,结果显示以粗酶催化可简化催化体系,并可将细胞催化体系所需高质量浓度蔗糖用量降至质量浓度5 g/100 mL,同时在不添加外源UDPG的条件下实现RD的高效生物催化,RA底物摩尔转化率达到93%,RD产量约为1?051?mg/L。     

转木糖还原酶基因XYL1酿酒酵母的构建及产木糖醇能力研究

将人工合成的树干毕赤酵母（Pichia stipitis）的木糖还原酶基因XYL1插入酿酒酵母（Saccharomyces cerevisiae）表达载体pYES2中,然后将重组质粒pYES2-XYL1导入酿酒酵母INVSc1中,构建转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1,最后采用营养缺陷培养基筛选转木糖还原酶基因酿酒酵母并对其产木糖醇的能力进行检测。结果表明,成功获得2株转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1-01、INVSc1/pYES2-XYL1-02,当两菌株以50 g/L木糖及10 g/L半乳糖为碳源发酵5 d后,木糖醇产量分别高达（13.68±2.37）g/L、（12.09±1.45）g/L,显著高于非转基因酿酒酵母INVSc1的木糖醇产量（1.08±0.37）g/L（P＜0.05）,说明XYL1基因的导入显著提高了酿酒酵母INVSc1生产木糖醇的能力（P＜0.05）。为采用基因工程酿酒酵母制备食用木糖醇提供了理论及技术基础。     

鲜食葡萄来源酵母菌的鉴定及其酿造学特性分析

为分析一株鲜食葡萄来源酵母菌YM7的类别及酿造学特性,采用形态学与分子生物学方法鉴定其种属,以商业化酿酒酵母（Saccharomyces cerevisiae）X16为对照,采用光密度法分析其生长特性和生理耐受性,对硝基苯基-β-D-吡喃葡萄糖苷显色法检测其β-葡萄糖苷酶合成能力,亚硫酸铋培养法检测其硫化氢产生能力,并与S. cerevisiae X16混合发酵葡萄汁,评价其对葡萄酒理化指标和香气特性的影响。结果表明,菌株YM7被鉴定为克鲁维毕赤酵母（Pichia kluyveri）,其生长性能、二氧化硫、柠檬酸耐受性与S. cerevisiae X16接近,葡萄糖耐受性低于S. cerevisiae X16,可耐受体积分数3%的乙醇,β-葡萄糖苷酶和硫化氢生产能力分别低于、高于S. cerevisiae X16。此外,与S. cerevisiae X16单独发酵葡萄酒相比,该菌株与S. cerevisiae X16混合发酵的葡萄酒挥发酸含量降低,香气化合物中酸类、醇类物质含量降低,酯类物质含量增加。     

柑橘皮渣生物转化燃料乙醇的研究进展

利用柑橘皮渣生物转化燃料乙醇是一种生产乙醇的安全可再生的方法。以柑橘皮渣为原料,通过酶或酸进行水解成可发酵糖液,利用糖酵解途径发酵乙醇的微生物将其转化为乙醇,提取脱水到99.5%浓度即得到燃料乙醇。这旨在解决柑橘产业大量副产物皮渣造成的环境污染资源浪费,同时生产乙醇作为枯竭的石油能源替代品。柑橘皮渣转化乙醇中,D-柠檬烯严重抑制微生物发酵,但合理控制各种影响因素可达到较高的乙醇产量。文中对柑橘皮渣生物转化燃料乙醇的过程及影响因素等进行了综述。