W／O微乳液中糖化酶的研究

在十六烷基三甲基溴化铵/正戊醇/异辛烷/水体系中,选取W/O型微乳液作为糖化酶的微环境,研究了其催化活性.并与其在水溶液中进行了比较.结果表明,在微乳液中,糖化酶催化淀粉水解反应的最佳反应温度和pH比在水溶液中低,反应的最大速度V_m和米氏常数K_m比其在水溶液中分别提高了近6倍和3倍.     

链霉菌A048产几丁质酶最佳发酵工艺研究

将链霉菌A048在完全培养基中培养至对数生长末期,离心洗涤收集菌丝体,然后接种入发酵产酶培养基中,进行二步发酵工艺牛产几丁质酶,几丁质酶活力比一步发酵工艺提高1．1倍,发酵周期共54h,比一步发酵工艺缩短66h;把菌丝体与几丁质粉共固定化,接入发酵产酶培养基中培养36h,几丁质酶活力比一步发酵工艺提高1．8倍,发酵周期缩短54h;在二步发酵工岂中另添加0．4％纤维素,几丁质酶活力可提高4倍,比一步发酵工艺提高10倍,酶活力达18．52U／mL。采用几丁质和纤维索双因子诱导二步发酵工艺可能是链霉菌A048生产几丁质酶的最佳工艺。     

棒状杆菌腈水合酶的形成条件

本文研究了棒状杆菌(cORYNEBACTERIUM)ZBB-2l腈水合酶形成的最适条件。在培养基中加入Fe2+、维生素B1和L-谷氨酸等,并以n-丁腈做诱导物,可明显促进该菌腈水合酶的生物合成。ZBB-21菌在选定的培养基中,于28℃培养64小时,其腈水合酶比活力可达83．1u／mg,而酰胺酶的比活力只有1．1u／mg。腈水合酶比活力比以前报道的提高9倍。     

漆酶／介体体系降解黄麻纤维木质素的研究

为了研究漆酶／介体处理过程中,黄麻纤维木质素结构的变化,采用二氧六环水溶液抽提制取了黄麻纤维木质素,再用漆酶／介体对其处理,通过GPC、元素分析、酚羟基含量测定、红外光谱以及核磁共振氢谱分析了漆酶／介体处理后,黄麻纤维木质素结构的变化。结果表明：经漆酶／介体处理后,黄麻纤维木质素重均分子量和数均分子量减小,酚羟基、醇羟基以及甲氧基含量降低,羰基含量增加。     

灌浆期高温和水分逆境对冬小麦籽粒蛋白质和淀粉含量的影响

灌浆期高温和水分逆境是影响小麦籽粒产量和品质的关键气候因子。以扬麦9号、徐州26和豫麦34三个小麦品种为材料,利用人工气候室模拟灌浆期高温和水分胁迫环境,研究了花后高温及温度和水分互作对小麦籽粒蛋白质和淀粉形成的影响。结果表明,高温显著提高了小麦籽粒蛋白质含量及清蛋白、球蛋白和醇溶蛋白含量,但降低了谷蛋白含量,导致麦谷蛋白／醇溶蛋白比值降低。高温显著降低了籽粒总淀粉和支链淀粉含量及支／直比。籽粒蛋白质和淀粉及其组分形成所需的适宜昼夜温差随小麦品质类型而异,但温度水平对籽粒蛋白质和淀粉的影响较温差大。在高温和水分逆境下,温度对籽粒蛋白质和淀粉含量的影响较水分逆境大,且存在显著的互作效应。小麦籽粒蛋白质含量均表现为干旱〉对照〉渍水,以高温干旱最高,适温渍水最低;淀粉含量为对照〉干旱〉渍水,以适温对照最高,而高温渍水最低。高温和水分逆境显著提高了籽粒醇溶蛋白含量而降低了谷蛋白含量及支链淀粉含量,使蛋白质谷／醇比和淀粉支／直比降低,以高温渍水对籽粒蛋白质和淀粉组分的影响最为显著。不同品种之间,高蛋白小麦籽粒蛋白质和组分的形成受高温和水分逆境的影响更大,而低蛋白品种籽粒淀粉形成显著受温度和水分逆境的调节。分析表明,在高温和水分逆境下籽粒蛋白质含量与清蛋白和醇溶蛋白显著正相关,籽粒淀粉含量与谷蛋白、支链淀粉含量及支／直比显著正相关。     

激光诱变选育高产PUFAs真菌菌株的研究

采用PIV400型Nd：YAG激光器,对少见报道的产PUFAs丝状真菌菌株Monoblepharis polymorpha Comu进行激光诱变育种。在激光照射剂量：波长532nm,功率50mW,时间10min下,孢子致死率为75％-80％。突变株中w-3系列PUFAs正突变率大于80％,w-3系列突变株FPAm59传代10代以上性能稳定,在土豆基质上摇瓶培养6d后,生物量达到8g/L,产油率53．4％,GLA343．1mg／L、从1209．0mg／L、EPA125．1mg／L、DHA119．4mg／L;分别比对照菌株提高了2．5倍、9．7倍、18．0倍、6．6倍、10．3倍、3．8倍。结果表明,激光诱变育种是获得PUFAs高产菌株尤其是生产w-3系列PUFAs菌株的有效方法。     

优质蛋白玉米胚乳贮存蛋白积累的电泳分析

玉米胚乳的２２ ｋＤ和２０ ｋＤ醇溶蛋白在授粉后１５ 天开始积累,编码２２ ｋＤ 和２０ ｋＤ醇溶蛋白的结构基因在胚乳发育过程中同时表达。优质蛋白玉米和ｏ２ 玉米的胚乳中,２２ ｋＤ和２０ ｋＤ醇溶蛋白的合成受到抑制,即ｏ２ 基因对２２ ｋＤ和２０ ｋＤ醇溶蛋白的合成有负的调节作用。Ｍｏ１７／ｏ２ 和Ｍｏ１７ 胚乳醇溶蛋白的双向电泳结果表明,Ｍｏ１７／ｏ２ 的２７ ｋＤ、２２ ｋＤ、２０ ＫＤ和１５ ｋＤ醇溶蛋白的合成均受到强烈的抑制。遗系０４１／ｏ２ 和遗系０４０／ｏ２ 胚乳醇溶蛋白的双向电泳结果表明,二者只在高分子量的蛋白质斑点区域有一些细微的差别。可溶性蛋白的ＳＤＳ－ＰＡＧＥ分析表明,Ｍｏ１７／ｏ２ 胚乳的可溶性蛋白比其同型系Ｍｏ１７ 少３８．７ ｋＤ 和２６．７ ｋＤ两条谱带,多２７．２ ｋＤ和２６．１ ｋＤ两条谱带。二者出现的可溶性蛋白的差异是ｏ２ 基因调控的结果。遗系０４１／ｏ２ 胚乳的可溶性蛋白比其同型系０４０／ｏ２ 多１８．６ ｋＤ和１７．６ ｋＤ两条谱带,少４０．２ ｋＤ 一条谱带,这与ｏ２ 基因修饰因子的作用密切关联     

烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌YPl产胞外蛋白酶的影响

考察了添加5％（V／V）浓度的正庚烷、正辛烷、正癸烷、十二烷、十四烷、十六烷等烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌（Bacillus licheniformis）YPl的生长及产胞外蛋白酶的影响。结果表明5％（v／v）浓度的各种烷烃瘩剂对YPl蛋白酶的稳定性及菌体生物量均无显著影响,正庚烷、正辛烷、正癸烷等溶剂显著抑制YPl产蛋白酶,而十二烷、十四烷、十六烷能提高YPI产蛋白酶1倍以上。发酵液中十四烷的浓度（1％-8％,V/V与蛋白酶的活力呈正相关性,添加十四烷后发酵过程中蛋白酶活力的显著增加出现在菌体生长的对数后期。培养过程中添加十四烷能导致YPl菌体形态显著变小。首次报道了烷烃溶剂对极端微生物产蛋白酶的影响。     

大肠杆菌脯氨酸基因的体内克隆及表达

用含mini－Mu的多拷贝质粒pEG5005体内克隆了大肠杆菌脯氨酸基因ProA⁺B⁺,其Pr0^＋克隆频率为1．46×1O^-3／Kan r转导子。遗传和生化分析表明这些克隆的Pr0^＋基团位于质粒上,用生长谱法对若干克隆子作了脯氨酸产量测定,但未检测出脯氨酸的积累。通过细胞内诱变Pro^＋克隆pPR3获得的去反馈抑制突变质粒pPR7,在脯氨酸Pr0AB基因缺失的寄主中可积累0．35mg／ml的脯氨酸,将此克隆转移至产1mg／ml脑氨酸的受体菌,其产酸量达2．5mg／ml,比受体高2．5倍,比供体提高了7倍,文中还对pEG5005和克隆pPR3作了内切酶分析。     

广藿香毛状根多倍体诱导及其植株再生

为了提高药用植物广藿香的次生物质广藿香醇含量,采用秋水仙素人工诱导染色体加倍技术,进行了广藿香毛状根多倍体诱导及其植株再生、倍性鉴定和挥发油组分广藿香醇含量的测定。结果表明,广藿香毛状根多倍体诱导的最佳条件为0.05%秋水仙素处理36 h,其多倍体诱导率可达40%以上;经秋水仙素加倍的广藿香毛状根在MS+6-BA 0.2 mg/L+NAA 0.1 mg/L培养基中培养60 d后可获得毛状根多倍体再生植株。与对照(二倍体植株)相比,广藿香毛状根多倍体再生植株根系更发达、茎更粗、节间变短、叶片的长度、宽度和厚度均较二倍体明显增大。根尖细胞染色体压片观察证实,所获得的广藿香毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为128;同时,其叶片的气孔保卫细胞体积及其叶绿体数目均约为对照的两倍;但其气孔密度则随着倍性增加而下降,二倍体植株叶片的气孔密度约为四倍体植株叶片的1.67倍。GC-MS测定结果表明,广藿香毛状根多倍体再生植株的广藿香挥发油组分广藿香醇的含量为4.25 mg/g干重,约为二倍体植株的2.30倍。该结果证实毛状根多倍体化可提高药用植物广藿香的广藿香醇含量。     

Synthesis and application of amphoteric starch graft polymer

An amphoteric starch-graft-polyacrylamide (S-g-PAM) was prepared by inverse emulsion polymerization, subsequent hydrolysis reaction and Mannich reaction. The copolymerization was carried out using ammonium persulfate and urea as redox initiator. The reaction conditions and application as flocculant were investigated. Experiments showed that in hydrolysis reaction, a stable emulsion of anionic S-g-PAM with high hydrolysis degree could be obtained in a shorter time when sodium carbonate and sodium hydroxide were used together as hydrolyzing agents. In Mannich reaction, after pre-formation of an aldehyde–amine adduct was added to the anionic emulsion product, the amination degree of amphoteric S-g-PAM could reach 43.6% and the highest solution viscosity was obtained. The application test showed that the results of treatment of several kinds of industrial waste water by amphoteric S-g-PAM were better than those treated with cationic polyacrylamide (PAM), hydrolytic PAM and amphoteric PAM.     

Starch Measurement in Plant Tissue Using Enzymatic Hydrolysis

This work explored completeness of starch hydrolysis in situ in relation to degree of gelatinization, starch content of tissue, evailable enzyme activity, and time allowed for hydrolysis. Maximum hydrolysis of starch in lyophilized red oak (Quercus rubra L.) root tissue with purified Diazyme (amyloglucosidase) or Clarase (Takadiastase) required high enzyme activity (2.4 U Diazyme or 48 U Clarase per mg starch). Results suggested that at least 70 U Clarase or 5 U Diazyme should be used per mg starch in routine analyses. Neither prolonging gelatinization (more than 15 min) nor hydrolysis (more than 24 to 48 lh) offset inadequate starch hydrolysis caused by insufficient enzyme activity. Starch was completely hydrolyzed in situ after 48 h without gelatinization by 5 U of Diazyme per mg starch. Tissue weight (5 to 100 mg) had no effect on starch hydrolysis by sufficient enzyme. Methanol: chloroform: water (12:5:3 by volume) freed tissues of solubles before starch hydrolysis. No interference with the glucose oxidase analysis of hydrolysates was encountered. In addition, the pigment free methanol–water fractions (soluble sugars, amino acids, organic acids) and chloroform fractions (lipids and pigments) were available or further analysis. Results obtained with red oak were verified with issue from other species such as jack pine (Pinus banksiana lamb.) and white spruce (Picea glauca (Moench) Voss). The resulting technique simply and reliably measured less than 5% starch in 5 mg lyophilized tissue, with a minimum of sample manipulation.     

Increasing the efficiency of the enzymatic hydrolysis of potato starch in a membrane reactor

The aim of the work was to define the physicochemical parameters of a reaction system that alter the effectiveness of a continuous recycle membrane reactor during potato starch hydrolysis. The enzymatic hydrolysis of starch in an ultrafiltration reaction system proceeded with a continuous decrease in the permeate flux, accompanied by an increase in dry substance content in both the permeate and retentate fractions. The decrease in the permeate flux was caused by an increase in feed viscosity. If a prehydrolysis process was conducted, it was possible to enzymatically hydrolyse potato starch in solutions with concentrations up to 20%. A quasi-steady state of starch enzymatic hydrolysis was reached in the ultrafiltration reaction system by alternately supplementing it with starch solution and water.     

有机溶剂可溶的超氧化物歧化酶的制备及其性质

本文报道用谷氨酸、十二醇、葡萄糖酸内酯合成了一种精脂（２Ｃ（１２）ＧＥ）,并制备了ＳＯＤ－糖脂复合体．所得的ＳＯＤ－糖脂复合体是脂溶性的而不是水溶性的,它在乙醇等有机溶剂中的活性比在水中高,而且存在一最适有机溶剂浓度。其对温度、ｐＨ、蛋白酶水解的稳定性比天然ＳＯＤ明显增强。     

Preparation of crystalline starch nanoparticles using cold acid hydrolysis and ultrasonication

Waxy maize starch in an aqueous sulfuric acid solution (3.16 M, 14.7% solids) was hydrolyzed for 2–6 days, either isothermally at 40 °C or 4 °C, or at cycled temperatures of 4 and 40 °C (1 day each). The starch hydrolyzates were recovered as precipitates after centrifuging the dispersion (10,000 rpm, 10 min). The yield of starch hydrolyzates depended on the hydrolysis temperature and time, which varied from 6.8% to 78%. The starch hydrolyzed at 40 °C or 4/40 °C exhibited increased crystallinity determined by X-ray diffraction analysis, but melted in broader temperature range (from 60 °C to 110 °C). However, the starch hydrolyzed at 4 °C displayed the crystallinity and melting endotherm similar to those of native starch. The starch hydrolyzates recovered by centrifugation were re-dispersed in water (15% solids), and the dispersion was treated by an ultrasonic treatment (60% amplitude, 3 min). The ultrasonication effectively fragmented the starch hydrolyzates to nanoparticles. The hydrolyzates obtained after 6 days of hydrolysis were more resistant to the ultrasonication than those after 2 or 4 days, regardless of hydrolysis temperatures. The starch nanoparticles could be prepared with high yield (78%) and crystallinity by 4 °C hydrolysis for 6 days followed by ultrasonication. Scanning electron microscopy revealed that the starch nanoparticles had globular shapes with diameters ranging from 50 to 90 nm.     

From water‐in‐oil to oil‐in‐water emulsions to optimize the production of fatty acids using ionic liquids in micellar systems

Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water‐in‐oil or oil‐in‐water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil‐in‐water emulsions), the imidazolium‐based IL acts as an enhancer of the lipase catalytic capacity, super‐activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)]. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1473–1480, 2015     

Lipase-catalyzed hydrolysis of fish oil in an optimum emulsion system

In this study, fish oil was hydrolyzed by lipase in a fish oil-in-water emulsion system in an effort to improve the functional properties of fish oil. Lipase activity was found to depend on the quality of the water/fish oil interface area. We selected several suitable emulsifiers, and their emulsifying activities were evaluated under a variety of conditions, including concentration, water-oil ratios, pH values, and temperature. Among the selected emulsifiers, the emulsifying activity of gelatin was higher than those of carboxymethyl chitin (CM-chitin), bovine serum albumin, and Tween-20, all of which are commercial emulsificers Moreover, the emulsifying activity of the gelatin solution was the highest at 0.5%, and was reduced with increasing concentrations of above 1%. The optimal water-oil ratio, pH, and temperature conditions were 40% (w/v), pH 8.0 and 40°C, respectively. Under these conditions, the emulsifying activity of gelatin solution was 86%. The emulsion structure of the gelatin solution was characterized by high density and small particle size. The degree of sardine oil hydrolysis in the emulsion system was 50% higher than that of the non-emulsion system. The lipid species of the lipase-prepared sardine oil hydrolysates were identified as triacylglycerol, 1,3- and 1,2-diacylglycerol, monoacylglycerol, and fatty acid.     

Physicochemical characterisation of enzymatically hydrolysed derivatives of acetylated starch

Potato starch modified to different degrees by substitution with acetyl groups was the subject of this study undertaken to determine the influence of conditions of enzymatic hydrolysis on the surface-active properties of hydrolysates of acetylated starch. The effect of acetylation of starch preparation on its susceptibility to enzymatic hydrolysis in the membrane reactor was also considered. All hydrolysates of acetylated starch samples investigated were found to bring a decrease in the surface/interfacial tension, both at the air/water and the toluene/water interfaces. For binary hydrolysate-surfactant systems, the surface mole fractions in the mixed adsorbed monolayer at the air/water interface were estimated. For mixed systems, the synergism in reducing the surface tension at the air/water interface was observed. The experimentally obtained dynamic surface tension data for the aqueous solution of acetylated starch hydrolysates were used to estimate the diffusion coefficients. Particle size distributions of the hydrolysates formed in the aqueous solutions were compared to those of commercial maltodextrin.     

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.     

A quantitative assessment of the importance of barley seed alpha-amylase, beta-amylase, debranching enzyme, and alpha-glucosidase in starch degradation.

Extracts of germinated barley (Hordeum vulgare L.) seeds of 41 different genotypes were analyzed for their activities of alpha-amylase, beta-amylase, alpha-glucosidase, and debranching enzyme and for their abilities to hydrolyze boiled soluble starch, nonboiled soluble starch, and starch granules extracted from barley seeds with water. Linear correlation analysis, used to quantitate the interactions between the seven parameters, revealed that boiled soluble starch was not a good substrate for predicting activities of enzymes functioning in in vivo starch hydrolysis as the extracts' abilities to hydrolyze boiled soluble starch was not correlated with their abilities to hydrolyze native starch granules. Activities of alpha-amylase and alpha-glucosidase were positively and significantly correlated with the seed extracts' abilities to hydrolyze all three starches. beta-Amylase was only significantly correlated with hydrolysis of boiled soluble starch. No significant correlations existed between debranching enzyme activity and hydrolysis of any of the three starches. Interactions between the four enzymes as they functioned together to hydrolyze the three types of starch were evaluated by path coefficient analysis. alpha-Amylase contributed to hydrolyses of all three starches primarily by its direct effect (noninteractive component). This direct contribution increased as the substrate progressed from the completely artificial boiled soluble starch, to the most physiologically significant substrate, native starch granules. alpha-Glucosidase contributed to the hydrolysis of boiled soluble starch primarily by its direct effect (noninteractive) yet contributed to starch granule hydrolysis primarily via its interaction with alpha-amylase (indirect effect). The contribution of beta-amylase to hydrolysis of boiled soluble starch was direct and it did not contribute significantly to hydrolysis of native starch granules.