低聚木糖生产用木聚糖酶的选择性合成

以里氏木霉(Trichoderma reesei)Rut C-30为产酶菌,研究了碳源、碳氮比对木聚糖酶酶系组成的影响.低分子量组分较多的木聚糖有利于促进内切-β-木聚糖酶的合成,酶解产物中低聚木糖的含量较高(80.70%).低碳氮比有利于促进内切-β-木聚糖酶的合成,抑制外切-β-木糖苷酶的合成.以低分子量较多的木聚糖(7g/L)为碳源,降低培养基的碳氮比为4.0,调控培养60h,用该木聚糖酶酶解粗木聚糖,产物中低聚木糖占总糖的86.32%.     

-木糖苷酶的分离纯化、酶学性质及水解机理研究

通过硫酸铵沉淀、超滤脱盐、离子交换层析、凝胶过滤层析等手段,从里氏木霉(Trichoderma reesei)Rut C-30木聚糖酶粗酶液中分离纯化得到分子质量110.8 ku、比活力为61.99 IU/mg的电泳纯β-木糖苷酶。酶学性质研究结果表明:该酶反应以温度60 ℃、pH值3.5为宜,在60 ℃以下、pH值3.0～8.0酶活性稳定,且该酶作用于对硝基苯基-β-D-木糖苷的K_m值和V_max值分别为0.29 μmol/mL和169.99 IU/mg。酶水解机理研究结果表明,该酶从低聚木糖的非还原性末端切断β-1,4-糖苷键释放出木糖,其最适作用底物是短链的低聚木糖,随着低聚木糖碳链的增长,酶作用效率逐渐下降,而对木聚糖几乎不降解。     

木聚糖酶最适pH值的研究

研究了以里氏木霉RutC - 3 0合成的木聚糖酶的最适pH值范围。结果表明 ,内切—木聚糖酶的最适pH值范围在 4.0～ 5 .0之间 ,β—木糖苷酶的最适pH值范围在 3 .0～ 4.0之间。研究还表明 ,与内切—木聚糖酶相比 ,pH值对β—木糖苷酶的影响更大。当pH值为 4.0时 ,酶水解总糖得率最高 ,适于制备木糖 ,而当pH值为 5 .0～ 6 .0时 ,较适于制备木低聚糖。     

低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状

对低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状进行了较全面的总结和评述。系统地介绍了里氏木霉木聚糖酶的的多样性以及碳源、pH和碳氮比等培养条件对合成内切木聚糖酶和木糖苷酶的影响,提出了调控这些培养条件选择性合成低木糖苷酶活的木聚糖酶的方法。     

地衣芽孢杆菌中性β-甘露聚糖酶的分离纯化

采用絮凝、超滤及离子交换对地衣芽孢杆菌中性β-甘露聚糖酶进行了分离纯化,纯化倍数为33倍,酶活收率达42%,所得纯化的β-甘露聚糖酶比活为4341 U/mg蛋白质.本文提出的分离纯化工艺易于工业放大.     

低聚合度木聚糖对木聚糖酶合成的影响

研究了木聚糖的聚合度和添加黄豆粉对里氏木霉合成木聚糖酶的影响,并以纯化木聚糖酶水解木聚糖。研究结果表明:木聚糖经酶水解后平均聚合度降低54%,戊聚糖含量为75.4%。采用低聚合度木聚糖为底物碳源和添加黄豆粉都可提高产酶效果。以12g/L低聚合度木聚糖添加2g/L黄豆粉,培养3d后木聚糖酶活力可达到113IU/mL,提高49.3%。通过对木聚糖酶进行纯化处理可以有效除去β 木糖苷酶。以体积分数10%的木聚糖酶水解35g/L木聚糖3h后,低聚木糖得率达到35.5%,而木糖得率仅为1.5%,低聚木糖与总糖的比值达到95.8%,随着酶解时间的延长,低聚木糖不会被降解。     

低聚合度木聚糖对木聚糖酶合成的影响

研究了木聚糖的聚合度和添加黄豆粉对里氏木霉合成木聚糖酶的影响，并以纯化木聚糖酶水解木聚糖。研究结果表明：木聚糖经酶水解后平均聚合度降低54％，戊聚糖含量为75．4％。采用低聚合度木聚糖为底物碳源和添加黄豆粉都可提高产酶效果。以12g／L低聚合度木聚糖添加2g／L黄豆粉，培养3d后木聚糖酶活力可达到113IU／mL，提高49．3％。通过对木聚糖酶进行纯化处理可以有效除去β-木糖苷酶。以体积分数10％的木聚糖酶水解35g／L木聚糖3h后，低聚木糖得率达到35．5％，而木糖得率仅为1．5％，低聚木糖与总糖的比值达到95．8％，随着酶解时间的延长，低聚木糖不会被降解。     

粗糙脉孢菌(Neurospora crassa)木聚糖酶的合成及性质研究

粗糙脉孢菌1602(Neurosporacrassa)可以产生木聚糖酶。以不同的植物纤维物料及纯木聚糖作为底物都可诱导产生较高的木聚糖酶活,其中玉米芯粉的诱导能力最强,酶活可达25.02IU/mL。纤维素与木聚糖混合对木聚糖酶合成具有促进作用。葡萄糖、木糖对木聚糖酶的合成有很强的阻遏作用,葡萄糖的阻遏作用大于木糖的阻遏作用。其木聚糖酶最适作用pH值为5.4,最适作用温度为60℃。     

β-半乳糖苷酶产生菌的筛选和发酵条件优化

以X-gal为显色剂,从大连三寰乳制品厂区周围土壤中筛选到15株β-半乳糖苷酶产生菌,以ONPG为底物,测定该15株菌液体发酵后的β-半乳糖苷酶活力,复筛到1株β-半乳糖苷酶高产菌株MFY-03。对菌株MFY-03摇瓶发酵产β-半乳糖苷酶的工艺条件进行了优化,确定了发酵培养基的组成为:葡萄糖1.0%,乳糖1.0%,酵母粉0.5%,蛋白胨1.0%,吐温80 1.0%,pH 6.8,于45℃、200 r/min条件下培养24 h,β-半乳糖苷酶活力达到3.74 U/mL。     

黑曲霉产β-葡萄糖苷酶发酵条件的研究

通过单因素及正交实验对现存黑曲霉菌株产酶发酵条件进行了研究,结果表明:当培养基为:玉米芯4.0%;酵母粉1.0%;KH2PO40.4%;MgSO40.08%;(NH4)2SO40.4%;培养温度30℃,摇床转速220r/min,接种量6.0%,装液量40mL/250mL,初始pH值5.0,发酵周期168h;此条件下β-葡萄糖苷酶酶活达56.96IU/mL。     

核糖核酸酶A的错流超滤复性

针对重组蛋白高表达形成的包涵体,基于中空纤维膜的错流超滤复性具有较大的应用潜力。为研究不同条件对基于中空纤维膜的错流超滤复性的影响,以核糖核酸酶A (RNase A)为例,以复性过程的活性收率和质量收率作为考察指标,设计了RNase A复性初始浓度(A)、跨膜压力(B)、循环流速(C)的3因素3水平正交实验。结果表明,错流超滤复性过程中,以上复性条件对RNase A的质量收率基本没有影响,对RNase A的活性收率具有显著影响,其较优组合为A1B1C2,即RNase A复性初始浓度为0.3 mg/mL、跨膜压力34.0 kPa、循环流速935 cm/min。在以上复性条件下,RNase A的活性收率可达92.31%,质量收率为77.56%。     

Expression of an Endo-β-1,4-glucanase Gene from Orpinomyces PC-2 in Pichia pastoris

The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.     

Thermal stabilization by polyols of β-xylanase from Bacillus amyloliquefaciens

Purified endo-β-1,4-xylanase of Bacillus amyloliquefaciens MIR 32 retained 100% of its activity after 4 days of incubation at 50°C. Sorbitol (400 mg cm⁻³) produced a 63-fold increase in the half-life of the enzyme at 65°C, which was only 29 min at this temperature in the absence of the polyol. This thermal stabilizing activity increased exponentially in respect to sorbitol concentration in the range 250–400 mg cm⁻³ and was dependent on the pH, showing a maximum at pH values between 5·25 and 8·0. The circular dichroism (CD) thermal scanning profile (50°C h⁻¹) at 224 nm showed that changes in the secondary structure of xylanase started at 65°C, while in the presence of sorbitol (400 mg cm⁻³) these modifications started at 80°C. This study indicated that sorbitol might be a valuable stabilizer for the use of β-xylanase from B. amyloliquefaciens at high temperatures. © 1998 SCI     

Improved extraction of phytochemicals from rooibos with enzyme treatment

An increasing demand for natural flavonoids necessitates the development of new technologies to improve the extraction of phytochemicals whilst retaining their bioactivity. Extracts from rooibos (Aspalathus linearis), well known as an herbal tea and food and beverage supplement, have shown significant potential for the treatment of various lifestyle conditions, but poor extraction of soluble matter limits its potential value and put limited supplies under pressure. This study demonstrates that pre-treatment with food-grade cellulase, ferulic acid esterase and/or pectinase increased the soluble solid yield from green and fermented rooibos when preparing an infusion, whereas ferulic acid esterase and β-glucanase/β-xylanase increased the total polyphenol yield. A combination of β-glucanase/β-xylanase and pectinase increased the yield of soluble solids from fermented rooibos in industrial evaluations by 33%. Although the total polyphenol and antioxidant content was reduced due to the extraction of ‘inactive’ sugar moieties, the extract exceeded industry benchmarks for both quality parameters. Furthermore, a clear extract was produced with no detrimental impact on its sensory attributes. Although the enzyme treatment reduced the aspalathin content of the extract, there was a concomitant increase in its flavone counterpart, isoorientin, which has significant pharmaceutical value for the treatment of both types of diabetes mellitus.     

Ultrafilter Conditions for High‐Level Waste Sludge Processing

Abstract

Optimal filtration conditions were evaluated for the ultrafiltration process planned for pretreating high‐level waste (HLW) sludge in the Hanford Waste Treatment Plant. This sludge must be filtered in the pretreatment process to remove sodium and, consequently, reduce the number of canisters for storage. The evaluation, which was based on Hanford HLW slurry test data, was performed to identify the optimal pressure drop and crossflow velocity for filtration at both high and low solids loading. Results from this analysis indicate that the actual filtration rate achieved is relatively insensitive to these conditions under anticipated operating conditions. The maximum filter flux was obtained by adjusting the system control valve pressure to between 400 kPa and 650 kPa while the filter feed concentration increased from 5 wt% to 20 wt%. However, operating the system with a constant control‐valve pressure drop of 500 kPa resulted in a reduction of less than 1% in the average filter flux. Also, allowing the control valve pressure to swing as much as ±20% resulted in less than a 5% decrease in filter flux. This analysis indicates that a back pressure setting of 500 kPa±100 kPa will give effectively optimal results for the system of interest.     

Continuous hot pressurized fluids extraction of isoflavones and soyasaponins from defatted soybean flakes

This work examined the effect of pressure (413–4410 kPa), temperature (333–393 K), solvent flow rate (10–25 mL/min), ethanol-to-water ratio (0–95%) and the feed loading (80–450 g) on hot pressurized fluids extraction of isoflavones and soyasaponins from defatted soybean flakes. A four-factor Taguchi experimental design was employed to optimize operational conditions of these extractions. Experimental results indicated that 95% of isoflavones and 76% soyasaponins were recovered from defatted soybean flakes by using hot pressurized 80% ethanol extraction at 383 K, 551 kPa and 25 mL/min with a 80 g feed loading. The recovered isoflavones were further purified by a solid–liquid column packed with Amberlite XAD 16-HP resin. The content of isoflavones in the collected fraction could be enhanced from 1 to 58% by weight.     

Role of Pressure in Coking of Thin Films of Bitumen

An important step in the formation of product from feed in a fluidized‐bed coker is the evolution of product and coke from layers of vacuum residue on the surfaces of heated particles and from liquid inside agglomerates of liquid and solid. In the present study, the yield of coke from Athabasca vacuum residue was measured using a reactor based on rapid induction heating of thin films of liquid feed on the surface of pieces of Curie‐point alloy. This approach allowed measurement of the yield of coke at pressures from 101–652 kPa, temperatures of 503 and 530°C, and reaction times from 10 to 240 s. When the liquid was reacted in thin films of ca. 20 µm, the effects of temperature and pressure on coke yield were insignificant. As the film thickness was increased to 120 µm, the yield of coke increased at all conditions. The yield of coke from thicker films was only sensitive to total pressure at 503°C reaction temperature, when the pressure was increased from 377 kPa to 652 kPa. Observable bubbling due to cracking reactions during coking was suppressed by increasing pressure, and the transition from quiescent liquid to bubbling liquid increased from circa 26 µm at 101.3 kPa to 78 µm at 652 kPa at 503°C. The bubbling transition was much less sensitive to pressure at 530°C, falling in the range from 22 µm to 43 µm as pressure increased from 101.3 to 652 kPa. These results suggest that the most important effect of pressure will be on the physical behaviour of liquid feed, due to its impact on bubble evolution from liquid inside agglomerates of liquid and solid particles. Depending on the liquid/solid ratio in an agglomerate, the formation of bubbles inside such a structure would make it weaker and easier to disperse on the fluidized bed reactor.     

融合蛋白CR2-GDH固定化及不对称还原制备(S)-4-氯-3-羟基丁酸乙酯

比较介孔分子筛材料SBA-15、MCM-41、海藻酸钙、改性二氧化硅4种载体固定化融合蛋白CR2-GDH其酶固载量和酶活回收率,选择SBA-15为固定化载体。研究固定化条件对固定化融合酶量的影响以及固定化酶的稳定性,固定化酶在双相体系催化不对称还原反应。结果表明,在pH值为5.5、酶浓度为1.4mg/mL、反应1h条件下,固定化酶量为27.7mg/g。加入25mmol/L的Ca²⁺,固定化酶的酶活回收率由58.6%提高到78.1%。与游离酶相比,固定化酶的热稳定性显著提高,40℃条件下酶活回收率提高19.1%。固定化酶水相中反复使用7批次后,剩余活性仍超过30%,具有较好的操作稳定性。与游离酶相比,固定化酶更耐受烷烃类有机溶剂。在水/有机溶剂双相反应体系中,Ca²⁺/SBA-15固定化酶和游离酶催化相比,产物得率提高23.8%。     

Production of biocatalysts on the basis of recombinant heterologous xylanase producer strains in the Penicillium verruculosum fungus: Their application in the hydrolysis of timber and wood processing industry wastes

The aim of this work was to create biocatalysts with an increased heterologous expression of endo-β-1,4-xylanase of P. canescens using recombinant P. verruculosum strains, to analyze the properties of new enzyme preparations, and to study the saccharifying activity of these preparations in the hydrolysis of plant raw materials, such as hogged aspen and detarred pine wood wastes of the timber and wood processing industries. The xylanase activity of the existing enzymatic preparations is insufficiently high to hydrolyze a xylan-rich biomass. The creation of increasingly xylanolytically active P. verruculosum-based recombinant strains containing homologous or heterologous genes of xylanase and mannanase is therefore a problem of great interest. Using the methods of genetic engineering, we obtained enzymatic preparations that are biocatalysts for the hydrolysis of plant raw material wastes of the sawmilling and wood processing industries and, according to the data of chromatographic fractionation, have compositions of 45–60% cellulase and 20–50% xylanase (which is optimal for the saccharifying of bagasse, along with aspen and pine wood). The originality of our technique lies in the creation of biocatalysts with predetermined properties, thus reducing appreciably the cost of enzyme preparation by eliminating the need to mix components of the carbohydrase complex for the hydrolysis of plant raw materials, e.g., aspen and pine wood.     

PERFORMANCE CHARACTERISTICS OF A CERAMIC MEMBRANE SYSTEM FOR MICROFILTRATION OF CORN STARCH HYDROLYSATE

A ceramic microfiltration membrane was used for the clarification of corn starch hydrolysate, having a dextrose equivalence of 95, to study the effect of process variables (transmembrane pressure, cross-flow velocity, and feed concentration) on permeate flux. Flux increased with increased cross-flow velocity for all transmembrane pressures and feed concentrations up to a volume concentration ratio of 100. Flux became asymptotic at pressures of 200-375kPa, indicating that microfiltration performance was limited by concentration-polarization. The optimum transmembrane pressure was higher at higher cross-flow velocities. A process model based on the resistances-in-series concept adequately described the observed variation of permeate flux with process variables such as transmembrane pressure, cross-flow velocity and feed concentration. Resistance due to concentration polarization decreased linearly with increase in cross-flow velocities for all feed concentrations, while fouling resistance increased linearly with increase in feed concentration.