Addition of protective agents for enhancing storage stabilization of alkaline polygalacturonate lyase

保藏稳定性对于商品酶具有重要的实际意义.通过对碱性果胶酶的保护剂进行研究,最终筛选得到最优复合保护剂配方为:Tween 20 1 mL/L,六偏磷酸钠1.5g/L,明胶5g/L,甘油5％(v/v),CaCl2 2 g/L;热稳定性提高18倍.此外在常温保藏时,在酶液中添加0.3‰山梨酸钾和0.3‰苯甲酸钠,能有效提高保藏效果,减少酶液染菌、混浊和发臭,最终常温保藏90d后酶活保留率为84.5％,达到工业化保藏的要求.     

酿酒酵母培养基中主要因素对海藻糖积累的影响

在对实验室保藏菌种Saccharomyces cerevisiae HY01优化前的摇瓶发酵观察的基础上,采用单因子与响应曲面相结合的实验设计方法研究了酿酒酵母培养基中的初始葡萄糖浓度、无机氮源硫酸铵浓度、酵母浸出粉浓度、碳源与无饥氮源的交互作用以及无机盐和微量元素浓度对海藻糖积累的影响。实验证明碳氮源交互作用明显,并得到了以上各因素的最优值,即初始葡萄糖浓度为20g／L,(NH4)2SO4浓度为4．5g／L,酵母浸出粉浓度为5g／L,微量元素溶液5．0ml／L;硫酸镁0．3g／L;磷酸氢二钠3．67g／L;磷酸二氢钾0．76g／L时,酿酒酵母中海藻糖的干重含量可以达到14．74％,比优化前海藻糖的干重含量12．24％提高了20．41％．     

手性拆分环氧氯丙烷菌株的筛选、鉴定及产酶条件研究

从土壤中筛选到5株环氧化物水解酶生产菌,并通过ITS序列鉴定了其中的C375菌,结果为黑曲霉（Aspergillus nigerZJB-09103）。考察了培养基不同碳源、氮源、金属离子和pH等对产酶的影响,得到了较佳的培养基条件：淀粉16g／L,豆饼粉3g／L,蛋白胨3g／L,KH2PO4 0．4g／L,K2HPO4 0．8g／L,MgSO4 0．2g／L,ZnSO4 0．03g／L,pH6．5。采用优化后的培养基条件,酶活力达到156．1U／L,比优化前初始发酵培养条件下的酶活提高了252％,当环氧化物水解酶催化时间为10h时,（s）-环氧氯丙烷的对映体过量值（e．e．）可达99．0％。产率为18．6％。     

海藻糖生产过程中产酶发酵条件的研究

研究了产酶的培养基组分和比例以及最佳培养条件对微球菌生产麦芽寡糖基海藻糖合成酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶(MTHase)的影响,得到最优培养基组成为：葡萄糖2．0％,酵母膏2．0％,蛋白胨1．0％,磷酸氢二钾0．1％,硫酸镁0．05％;优化后的培养条件为：以15％的接种量接种至250mL的锥形瓶中,装液量为50mL,初始pH值7．5～8．5,培养温度为30℃,摇床培养4d。经优化后菌体干重由原来的1．938g／L增加到18．5g／L,生物量几乎增长了10倍;而酶活也由原来的30．64U／g增加到206．11U／g,酶活提高了接近7倍。     

利用Design-Expert软件优化丝氨酸羟甲基转移酶产酶培养基

利用Design-Expert软件中水平设计和响应面分析法对产酶基本培养基主要成分进行了优化,经过逐步回归分析建立了丝氨酸羟叫基转移酶（SHMT）活力对培养基主要成分的二次回归模型,其回归方程的决定系数达到了0．9984。得到的最佳培养琏主要组成为：葡萄糖29．5g／L、硫酸铵18．1g/L、玉米浆3．79g／L。SHMT活力最高达到113．7U／ml,比优化前（77U／mL）提高了47．7%。优化后的酶液经酶促反应50h,能催化产生10g／L的L-丝氨酸,比优化前（6g／L）提高66．7％。     

聚乙烯醇脱氢酶的提取及检测

为解决结合在细胞上的可溶性蛋白聚乙烯醇脱氢酶（PVADH）的检测困难问题,从提取及检测两方面对该酶进行研究,并对检测方法进行改进。结果表明,非离子型表面活性剂Triton X-100对可溶性蛋白PVADH的提取效果优于离子型表面活性剂炕基苯磺酸钠（LAS）和溴化十六烷基吡啶（CPB）,酶活力比LAS和CPB提取后所得酶活力分别提高246．5％和831．3％。而非离子型表面活性剂中,Triton X-100与Tween80相比,所得最高酶活提高了101．4％。Triton X—100浓度和提取时间对测定有明显影响,以1％Triton X-100提取18h为宜,最高比酶活达14．9U／g。在PVADH检测体系中,加入电子受体启动反应比加入酶液与底物启动反应可使酶活性分别提高60．6％和126．5％;酶液与吡咯喹啉醌（PQQ）预先保温对检测该酶活性是十分重要的,可使酶活性提高59．1％.在检测系统中加入的KCN、CaCl2和PQQ的适宜浓度分别为1．ommol／L、0．5mmol／L和2μmol／L,可使测定酶活分别提高37．1％、38．7％和214．0％．     

壳聚糖固定化木聚糖酶的研究

从青霉菌m8提取出木聚糖酶,将其固定在用戊二醛交联的壳聚糖载体上。1．0g壳聚糖与4％的二醛结合固定3．5mg蛋白,酶活回收率为46．6％。在酶的最适pH为4．6,固定化酶为pH3．8。原酶的最适温度为55℃,固定化酶在60－75℃都具有较高活性。固定化酶的耐热性优于原酶,固定化酶的表现Km值略低于原酶,前者为5．0×10－2g／L,后者为3．58×10－2g／L。     

益母草（Leonurus artemisia）灭螺效果研究

本实验研充分别采用益母草的根、茎、叶的0．10、0．25、0．50、2．00、1．00g／L和水苏碱的0．20、0．40、0．60、0．80、1．00g/L 5个不同浓度梯度的处理液处理钉螺,设清水和0．001g／L浓度的氯硝柳胺溶液为对照。结果表明：（1）益母草各部分的水浸液均有很好的灭螺效果。用不同浓度的处理液浸杀钉螺,在不同时间的处理下,钉螺死亡率存在差异,其钉螺死亡率是随处理浓度的增加和时间的延长呈上升趋势,0．5g／L以上的益母草根、茎、叶、化水浸液和浓度达0．60g／L以上的水苏碱处理液均可达到100％的明显毒杀钉螺致死效果,与通常使用浓度0．0001g／L氯硝柳胺溶液的灭螺效果相当,不过益母苹根、茎、叶水浸液的毒效较氯硝柳胺略慢,用0．0001g／L氯硝柳胺溶液处理钉螺2—3d可达100％的死亡率,而用0．5g／L以上的益母草根、茎、叶水浸液水溶液处理需要3—5d才能达到同样的效果;灭螺效果顺序依次为：叶〉茎〉根。（2）钉螺趋避性研究表明水苏碱和益母草根、茎和叶的处理液对钉螺具有明显的驱逐作用,而盐酸益母草碱几乎没有作用。由此获得化感作用植物益母草灭螺的化学生态学证据。为研制新的具中国特色的植物成份灭螺剂打下了基础。并为合成仿生灭螺剂以及最终构建生态工程中强化感作用植物群落灭螺提供理论依据。     

提高狗蔷薇离体培养植株再生频率

以狗蔷薇（Rosa canina Inermis）为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合,大幅度提高了狗蔷薇离体培养植株再生频率。结果表明,2．0mg／L 6-BA＋0．3mg／L 2,4-D的组合较为适宜,其不定芽再生率为87％,增殖率为3．0;而CPPU和2,4-D的适宜组合为1．5mg／L＋0．3mg／L,其不定芽再生率高达93％,增殖率为5．0。同时,研究结果显示,以MS＋40g／L蔗糖＋6．0g／L琼脂粉＋3．5mg／L AgNO3＋1．5mg／L CPPU＋0．1mg／L 2,4-D＋0．05mg／L GA3作增殖培养基效果最好,不定芽诱导率为89％,增殖率为5．5;利于生根的培养基为1／4MS＋20g／L蔗糖＋3．5g／L琼脂＋0．3％活性碳＋0．1mg／L IBA＋0．1mg／L NAA,生根率为91％。     

荧光素酶基因luc在大肠杆菌中表达条件优化

对重组荧光素酶大肠杆菌菌株M15／pQE30-luc进行了表达条件的优化研究。单因素结果表明：在初始pH值7．0,装液量为20％,2％的接种量,终浓度为0．5mmol／L的IPTG,添加10—30mmol／L的Mg^2＋,摇床转速为200r／min,37℃诱导3．5h酶的表达量最高。正交试验结果表明：初始pH值为7．0,添加40mmol／LMg^2=,接种量2％,装液量为20％时表达量最高,比酶活达1．63×10^8RFU／mg蛋白。     

液态型凝乳酶储藏条件的初步研究

为进一步延长液态型凝乳酶的储藏时间,以金属离子、糖类、多羟基醇、大分子助剂和表面活性剂等为稳定剂优化得到最优的复合保护剂配方:山梨醇7.5%、明胶1%、甘油15%、吐温400.25%、氯化钾50 mmol/L;添加0.1 g/L对羟基苯甲酸乙酯可有效抑制在储藏过程中由杂茵繁殖导致的发臭、浑浊。添加复合保护剂和防腐剂的液态型凝乳酶,室温放置90 d,凝乳酶活力保留率为80.8%,比对照组提高41.6%。     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

The effects of some storage conditions on viability of Lecanicillium lecanii conidia to whitefly (Homoptera: Trialeurodes vaporariorum)

A study on the survival of Lecanicillium lecanii conidia in storage at room temperature was carried out. Firstly, drying methods of conidia powder were compared. Vacuum-freeze drying (VFD) was more suitable for drying conidia as compared to vacuum drying (VD) at room temperature. Vacuum-freeze drying for 24-h resulted in a water content of 5.4%, and a viability, determined as germination of conidia in 2% glucose solution after16 h, was 90.3% and the infection in greenhouse whitefly, Trialeurodes vaporariorum was about 94.7% at a dose of 1×10⁸ conidia/mL. Secondly, the factors influencing viability of conidia stored at room temperature were evaluated in the laboratory. Temperature was the most critical factor influencing conidial storage stability, among the tested factors affecting survival of conidia stored at room temperature for 6 months. Both conidial germination and infection of hosts decreased with storage temperature increasing from 15 to 35°C, and at 35°C the survival of stored conidia for 6 months was near zero. The moisture content of the conidial powder was another major factor influencing viability of stored conidia at room temperature. Conidial powder dried to about 5% moisture content showed higher viability than non-dried conidial powder. For the carriers, clay and charcoal were more suitable for storage of L. lecanii conidia at room temperature. At a room temperature of 25°C, L. lecanii conidia which were dried to 5% water content and mixed with clay or charcoal could retain about 50% survival after 6 months' storage.     

保护剂对重组大肠杆菌NMN转移酶稳定性的影响

通过向NMN转移酶( Nmnat)中添加保护剂以提高其热稳定性及储存稳定性,扩大酶的使用范围。研究了固体醇类(山梨醇、甘露醇)、糖类(海藻酸钠、蔗糖、甘露糖)和有机溶剂( DMSO、丙二醇-单甲醚、乙醇、甘油)对Nmnat的稳定性的作用,通过正交实验得到一种复合保护剂,并研究复合保护剂对Nmnat最适反应温度、pH稳定性、储存稳定性的影响。结果表明,山梨醇、海藻酸钠和DMSO能够显著提高Nmnat的热稳定性。复合保护剂配方为山梨醇1.5 g/L,海藻酸钠1.0 g/L, DMSO 0.5%。复合保护剂的添加使Nmnat的最适反应温度从37℃提高到50℃;50℃保温2 h酶活提高了24.5%;pH使用范围由7.0~8.0扩大到6.0~8.0;4℃储存28 d后,酶活保留率提高了15.65%。     

Properties of 4-ene-5 alpha-reductase and studies on its solubilization from porcine testicular microsomes

The activity of 4-ene-5 alpha-reductase was assayed in porcine testis homogenates and subcellular fractions, using testosterone as substrate. 'Marker' enzyme activities were utilized to indicate the purity of the subcellular fractions. 4-Ene-5 alpha-reductase activity was associated with the microsomal fraction; there was no activity in the purified nuclear fraction. Enzyme activity was higher in the testes of 6 week old pigs than those of 3 and 17 week old animals, and a range of activity was found. The enzyme was unstable when stored at -20 degrees C but the addition of albumin (0.1%, w/v) or glycerol (20%, v/v) to the buffer and storage at -70 degrees C or in liquid nitrogen ensured that maximal activity was retained for at least 35 days. In addition to 5 alpha-DHT, other 5 alpha-reduced metabolites and 4-androstenedione were formed in this reaction; NADPH was the preferred cofactor, but 40% of the 4-ene-5 alpha-reductase activity was retained when NADH was used. Solubilization of the microsomal enzyme was achieved using sodium citrate (0.1 M); 4-ene-5 alpha-reductase activity was enhanced to greater than 120% and 60% of this activity was in the soluble fraction. The optimum pH and temperature for both soluble and membrane-bound 4-ene-5 alpha-reductase were 6.9 and 32 degrees C, respectively. The mean apparent Km and Vmax were 0.6 mumol/l and 158 pmol/min/mg microsomal protein for the microsomal enzyme and 1.42 mumol/l and 212.0 pmol/min/mg soluble protein for the solubilized 4-ene-5 alpha-reductase. The estimated sedimentation coefficient was 11.6.     

Vanadium containing bromoperoxidase: An example of an oxidoreductase with high operational stability in aqueous and organic media

The conversion is described of phenolsulphonephtalein (phenol red) to 3,3',5,5'-tetrabromophenolsulphonephthalein (bromophenol blue) by bromoper-oxidase from the brown alga Ascophyllum nodosum. This reaction provides a convenient assay for the detection of bromoperoxidase activity in vitro. Bromoperoxidase was shown to be stable under turnover conditions for three weeks at room temperature, catalyzing the bromination of phenol red into bromophenol blue. When stored at room temperature in organic sol vents such as acetone, methanol, ethanol [present up to 60% (v/v)], and 1-propanol [40% (v/v)], bromoperoxidase was stable for more than one month. As far as we know this is the first example of an oxidoreductase which displays such great stability. This enhances the applicability of the enzyme in organic synthesis.     

冷风干燥制备高活菌的恶臭假单胞菌菌粉

【目的】获得高活菌恶臭假单胞菌菌粉,提高菌体干燥及保藏存活率。【方法】选用冷风干燥法制备活菌粉,并优化吸附载体与保护剂。【结果】冷风干燥制备恶臭假单胞菌菌粉干燥存活率普遍达到65%以上,显著优于喷雾干燥(24%);对载体与保护剂进行正交试验优化,确定了载体为混合的硅藻土和碱处理玉米芯粉,混合比为1:2,保护剂(质量比)为甘露醇7%、谷氨酸钠5%、甘油1%,制得菌粉活菌数为1.03×1011 CFU/g,室温保藏30 d和4 °C保藏60 d存活率分别达到40.54%和71.67%。【结论】冷风干燥温度相对较低(10?40 °C),对菌体损伤小,碱处理玉米芯粉、甘露醇和谷氨酸钠是提高菌粉保藏存活率的重要因子,此法克服了革兰氏阴性菌菌粉不易制备和不耐保藏的瓶颈。     

Rapid purification of protein kinase C by high performance liquid chromatography

Protein kinase C was purified from rat brain cytosol by using a high performance liquid chromatography (HPLC), Pharmacia FPLC system. This procedure employed a column chromatography on DE-52, followed by three steps of HPLC procedures with threonine-Sepharose (prepared as described in this report), TSK gel Phenyl-5PW (Toyo Soda), and TSK gel G3000SW (Toyo Soda) columns. Starting from about 30 g of rat brain, approximately 200 micrograms of pure enzyme was obtained. The procedure was very simple and highly reproducible. The enzyme thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 10% (w/v) glycerol and 0.05% (w/v) Triton X-100, the enzyme could be stored at -80 degrees C for several months.     

Phytase from antarctic yeast strain Cryptococcus laurentii AL27

Cryptococcus laurentii strain AL(27) demonstrating significant potential for intracellular phytase production was selected by 2-step screening of Antarctic yeasts. The strain showed increased phytase activity in a culture medium with 40 g/L sucrose, KH(2)PO(4) providing 5 mg/L phosphorus, and cultivation temperature of 24 degrees C, which relates it to psychrotrophic microorganisms. The enzyme kinetic characteristics according to sodium phytate were K (m) = 0.98 mmol/L, v (lim) = 33.3 mumol g(-1) min(-1). The enzyme had maximum activity at 40 degrees C and acted within a wide pH range: from 2.0 to 5.5, which is of positive significance for its direct inclusion into the feed of monogastric animals.     

Oxygenation of unsaturated fatty acids in seeds during storage

Seeds of Cichorium intybus L., Crepis thomsonii Babc, and Crepis vesicaria L, were stored from 4 to 8 years at 5°C and then for 18 months under a variety of conditions. Oxygenated acids in Cichorium intybus oil increased from approximately 1% initially to 3% in the first storage period and to 17% while stored at room temperature during the second period. The corresponding levels at these three stages for Crepis thomsonii were 2, 6 and 18%. By gas chromatography (GC) and GC-mass spectrometry, the major oxygenated acids formed during storage were identified as hydroxy acids with conjugated unsaturation and 9,10-epoxy acids. In Crepis vesicaria seed, oil of which contained 53% vernolic (12,13-epoxy-9-octadecenoic) acid originally, approximately 2% of 9,10-epoxides were formed during the storage at room temperature. Levels of hydroxy acids with conjugated unsaturation in this species were 0.3% initially, 2% after 5 years at 5°C, and 9% after 18 months at room temperature. Primary substrates from which oxygenated acids were formed in the three species were crepenynic and linoleic acids, and the almost exclusive formation of 9,10-epoxide from linoleic acid indicated enzymatic involvement.