Biosorption of Cr (VI) with Trichoderma viride immobilized fungal biomass and cell free Ca-alginate beads

Ability of Cr (VI) biosorption with immobilized Trichoderma viride biomass and cell free Ca-alginate beads was studied in the present study. Biosorption efficiency in the powdered fungal biomass entrapped in polymeric matric of calcium alginate compared with cell free calcium alginate beads. Effect of pH, initial metal ion concentration, time and biomass dose on the Cr (VI) removal by immobilized and cell free Ca-alginate beads were also determined. Biosorption of Cr (VI) was pH dependent and the maximum adsorption was observed at pH 2.0. The adsorption equilibrium was reached in 90 min. The maximum adsorption capacity of 16.075 mgg(-1) was observed at dose 0.2 mg in 100 ml of Cr (VI) solution. The high value of kinetics rate constant Kad (3.73 x 10(-2)) with immobilized fungal biomass and (3.75 x 10(-2)) with cell free Ca- alginate beads showed that the sorption of Cr (VI) ions on immobilized biomass and cell free Ca-alginate beads followed pseudo first order kinetics. The experimental results were fitted satisfactory to the Langmuir and Freundlich isotherm models. The hydroxyl (-OH) and amino (-NH) functional groups were responsible in biosorption of Cr (VI) with fungal biomass spp. Trichoderma viride analysed using Fourier Transform Infrared (FTIR) Spectrometer.     

应用固定化里氏木霉糖化玉米秆纤维素的研究

采用多孔聚酯材料固定里氏木霉（TrichodermareeseiRutC30）菌丝细胞,将固定化细胞在生长限制条件下重复分批培养,使纤维酶的合成与玉米秆纤维原料的酶解糖化耦合在一个反应器中同时进行。在30℃、初始pH4.8、摇瓶转速150r／min的条件下,连续重复进行12次分批培养试验。每批玉米秆用量为60g／L,培养周期4．5d,共54d。培养液中含滤纸酶活力平均为0.70IU／ml,还原糖26．41g／L,糖化率达到理论值的89.11％。固定化菌丝形态正常,菌量保持在10g／L左右。在间歇添料条件下,玉米秆原料的总量可提高到120g／L,7d后还原糖浓度达52.81g／L,糖化率为89．20％。利用固定化里氏木霉同时产酶和糖化植物纤维原料,工艺简便、成本低廉、易于连续自动化操作,是一条有效利用可再生纤维素资源的新途径。     

Production of diacylglycerols from glycerol monooleate and ethyl oleate through free and immobilized lipase-catalyzed consecutive reactions

The ability of free and immobilized lipase on the production of diacylglycerols (DAG) by transesterification of glycerol monooleate (GMO) and ethyl oleate was investigated. Among three free lipases such as lipase G (Penicillium cyclopium), lipase AK (Pseudomonas fluorescens) and lipase PS (Pseudomonas cepacia), lipase PS exhibited the highest DAG productivity, and the DAG content gradually increased up to 24 hours reaction and then remained steady. The comparative result for DAG productivity between free lipase PS and immobilized lipases (lipase PS-D and Lipozyme RM IM) during nine times of 24 hours reaction indicated that total DAG production was higher in immobilized lipase PS-D (183.5mM) and Lipozyme RM IM (309.5mM) than free lipase PS (122.0mM) at the first reaction, and that the DAG production rate was reduced by consecutive reactions, in which more sn-1,3-DAG was synthesized than sn-1,2-DAG. During the consecutive reactions, the activity of lipase PS was relatively steady by showing similar DAG content, whereas DAG production of lipase PS-D and Lipozyme RM IM was gradually decreased to 69.9 and 167.1mM at 9th reaction, respectively, resulting in 62% and 46% reduced production when compared with 1st reaction. Interestingly, from 7th reaction lipase PS produced more DAG than immobilized lipase PS-D, and exhibited a stable activity for DAG production. Therefore, the present study suggested that DAG productivity between GMO and ethyl oleate was higher in immobilized lipases than free lipases, but the activity was reduced with repeated uses.     

Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-life of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.     

A simple method of cellulase immobilization on a modified silica support

Two types of cellulase (Robillarda sp. Y-20 and Trichoderma reesei) were immobilized on Aminosilica-1 by physical adsorption. Enzymes were quickly immobilized and stable on the support. Specific activities of two types of immobilized cellulase were 0.24 U/mg support with Robillarda cellulase and 0.15 U/mg support with the Trichoderma one as CMCase. The pH-activity curves of both cellulase shifted slightly to lower pH on immobilization. Both immobilized cellulases showed essentially the same pH stabilities as their free forms. However, the immobilized enzymes were less stable than the free forms at temperatures higher than 50°C.     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

固定化纤维二糖酶的研究

黑曲霉 (AspergillusnigerLORRE 0 12 )的孢子中富含纤维二糖酶 ,将这些孢子用海藻酸钙凝胶包埋后 ,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定 ,半衰期为 38d ,耐热性和适宜的pH范围均比固定化前有所增加 ,其Km 和Vmax值分别为 6 .0 1mmol L和 7.0 6mmol (min·L)。利用固定化纤维二糖酶重复分批酶解10g L的纤维二糖 ,连续 10批的酶解得率均可保持在 97%以上 ;采用连续酶解工艺 ,当稀释率为 0 .4h- 1 ,酶解得率可达 98.5 %。玉米芯经稀酸预处理后 ,其纤维残渣用里氏木霉 (Trichodermareesei)纤维素酶降解 ,酶解得率为6 9.5 % ;通过固定化纤维二糖酶的进一步作用 ,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除 ,酶解得率提高到 84.2 % ,还原糖中葡萄糖的比例由 5 3 .6 %升至 89.5 % ,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。     

Production of L-lysine by free and PVA-cryogel immobilizedCorynebacterium glutamicum cells

Summary Free and immobilized cells ofC. glutamicum were analyzed for production of L-lysine. Non-growing free cells as well as immobilized ones produced higher levels of lysine than growing free cells, when cultured for 24 h in a medium containing 80 g/L glucose as a carbon source. The effect of initial biomass and yeast extract concentrations was investigated in order to improve lysine production.     

Cunninghamella echinulata a new biosorbent of metal ions from polluted water in Egypt

Thirty-two fungal species were isolated from a polluted watercourse near the Talkha fertilizer plant, Mansoura Province, Egypt. Aspergillus niger, A. flavus, Cunninghamella echinulata and Trichoderma koningii were isolated frequently. On the basis of its frequency, Cunninghamella echinulata was chosen for biosorption studies. Free and immobilized biomass of C. echinulata sequestered ions in this decreasing sequence is: Pb >Cu >Zn. The effects of biomass concentration, pH and time of contact were investigated. The level of ion uptake rose with increasing biomass. The maximum uptake for lead (45 mg/g), copper (20 mg/g) and zinc (18.8 mg/g) occurred at 200 mg/L biomass. The uptake rose with increasing pH up to 4 in the case of Pb and 5 in the case of Cu and Zn. Maximum uptake for all metals was achieved after 15 min. Ion uptake followed the Langmuir adsorption model, permitting the calculation of maximum uptake and affinity coefficients. Treatment of C. echinulata biomass with NaOH improved biosorbent capacity, as did immobilization with alginate. Immobilized biomass could be regenerated readily by treatment with dilute HCl. The biomass-alginate complex efficiently removed Pb, Zn and Cu from polluted water samples. Therefore,Cunninghamella echinulata could be employed either in free or immobilized form as a biosorbent of metal ions in waste water.     

Lipid accumulation in Trichoderma species

Abstract Two filamentous fungi, Trichoderma harzianum and Trichoderma viride , were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient (C/N = 60) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride . In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. However the small quantity of biomass produced (2 g dry weight/l) limited the quantity of lipid obtained. Neutral lipids, free fatty acids and phospholipids were monitored during 8 days of cultivation of the two fungi.     

Enzymatic polymerization catalyzed by immobilized endoglucanase on gold

Enzymatic polymerization was carried out on gold by immobilized genetically engineered endoglucanase II (EGII) from Trichoderma viride , and the polymerization behavior and the produced cellulose were analyzed in comparison with those by free enzymes. Mutant EGIIs were EGII(core2) and EGII(core2H), which consist of two sequential catalytic core domains with one His-tag (His6) on N-terminal and with totally two His-tags on both terminals, respectively. His-tagged EGIIs were immobilized via Ni chelators of nitrilotriacetic acid (NTA) introduced on gold surface. From SPR measurements, the affinity of EGII(core2H) to the surface was higher than that of EGII(core2), and the molecular occupation area of EGII(core2H) was larger than that of EGII(core2), indicating that EGII(core2H) was immobilized with utilizing two His-tags introduced on both terminals. The hydrolytic activity of the immobilized EGII(core2H) using cellohexaose as substrate was slightly lower than that of free EGII(core2H) when they were compared at the same amount in the hydrolytic system. Enzymatic polymerization catalyzed by both immobilized EGII(core2) and EGII(core2H) proceeded with producing highly crystalline cellulose in comparison with free enzyme. Immobilization of the endoglucanase is thus effective to obtain crystalline cellulose due to the high density of the catalytic domain on gold.     

The use of bioluminescence stimulant on the immobilized strain, P. putida mt-2 KG1206, with toluene analog inducers and environmental samples

This study was conducted to investigate the applicability of the stimulant conditions for the bioluminescence activity of a recombinant strain of Pseudomonas putida, mt-2 KG1206, when immobilized using alginate polymer. The bioluminescence activity of the immobilized strain was generally approximately three to five times lower than the subcultured strain, and the activity was observed to slowly decrease. These facts may have been caused by several factors, such as the low biomass and the time required for diffusion into the entrapped biomass. Although different inducers produced different degrees of stimulation, immobilized bacteria modified with KNO₃ consistently produced more bioluminescence than those treated with sodium lactate, regardless of the inducer chemical tested. Cells treated with KNO₃ exhibited 2.8 times greater bioluminescence than that of the control activity. This condition also stimulated the bioluminescence activities of the immobilized bacteria exposed to contaminated groundwater samples. Based on these results, the immobilized KG1206 presented in this research can be used as a portable assay for the purpose of preliminary on-site monitoring of specific inducer contaminants, with subsequent off-site instrumental analysis, suggesting the potential of this immobilized cell for preliminary application in a field-ready bioassay.     

Characterization of microbial endo-β-glucanase immobilized in alginate beads

Endo-β-glucanase (endo-β-1,4-glucano-glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH-activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilizatíon. The enzyme immobilized in alginate beads showed a K_m value (1.02% w/v) lower than that of the enzyme (1.31%). The apparent V_max of immobilized cellulase preparations (238.3 μmol glucose/ml × h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60°C) of the free and entrapped enzymes remained unaltered. In contrast, the half-life of the endoglucanase immobilized in calciumalginate beads was 4.6 h at 55°C and 5.4 h at 60°C, while that of the free enzyme was 3.0 h at 55°C and 1.2 h at 60°C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and β-glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250–720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized β-glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co-immobilization of both types of enzymes reduced by hydrolytic efficiency to half.     

Continuous limonin degradation by immobilizedRhodococcus fascians cells in K-carrageenan

Limonin can be effectively degraded byRhodococcus fascians cells. These bacteria can be entraped in -carrageenan, and used in a continuous stirred tank reactor to degrade limonin in a continuous process. The effects of temperature limonin concentration, dilution rate, and aeration on the reactor behaviour have been tested, and the results correlated with changes in limonin conversion, substrate degradation rate, and free and immobilized biomass. Results showed that the immobilized cells were able to debitter limonin-containing media and the immobilized biomass was quite stable throughout the operational conditions tested. A population of free biomass was present in the reactor, the quantity of which was dependent on dilution rate. The immobilized bacteria increased its limonin-degrading capability when the substrate concentration was increased. The aeration was not strictly necessary for limonin degradation. Additionally, the immobilized cells were active and stable for more than 2 months of continuous operation, and were able to recover their limonin-degrading capability when used intermittently. Finally, none of the main components of a juice was noticeably altered during limonin degradation, so the reactor response was good enough to consider its application.     

固定化小球藻产氧及光合速率的研究

通过对固定化小球藻产氧及光合速率的研究,进一步解决水产养殖水体溶解氧不足的问题,为生物增氧技术投入使用奠定基础.固定化具有较高的生物量及生物活性,在研究中被广泛使用.运用固定化技术对固定化小球藻产氧及光合速率进行了研究,结果表明,固定化小球藻具有较好的生物量及生物活性,海藻酸钠+羧甲基纤维素钠+硅藻土作为固定化基质有利...     

Carbon dioxide fixation by immobilized chloroplasts

Chloroplasts of chinese mustard (Brassica campestris L.) were immobilized in polyacrylamide gel. A 8% polymer concentration was suitable for the immobilization. The activity of the carbon dioxide fixation of immobilized chloroplasts was 65% of that of free chloroplasts. The optimum conditions for the carbon dioxide fixation of immobilized chloroplasts were similar to that of native chloroplasts. However, immobilized chloroplasts were more stable under alkaline conditions and high temperatures than native chloroplasts. Light penetration of the gel was not a limiting parameter of the carbon dioxide fixation. The lifetime of immobilized chloroplasts was three times longer than that of free chloroplasts. 3-Phosphoglyceraldehyde and other compounds were produced continuously by immobilized chloroplasts.     

Properties of cellulase immobilized on agarose gel with spacer

Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated CH-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose.     

Production and immobilization of D-aminoacylase of Alcaligenes faecalis DA1 for optical resolution of N-acyl-DL-amino acids.

The production of D-aminoacylase by Alcaligenes faecalis DA1 was induced 5- to 50-fold by N-acetyl-D-amino acids. This strain produced about 443 units of D-aminoacylase and 52 units of L-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-DL-leucine as the carbon source. The D-aminoacylase was partially purified by Fractogel DEAE 650 column chromatography and then immobilized on another Fractogel DEAE 650 column. The catalytic activity of the immobilized D-aminoacylase was 2,650 units per milliliter of gel. The Km values for the free and the immobilized enzymes were found to be 1.00 and 0.22 mM, respectively, using N-acetyl-D-methionine as a substrate. The optimal reaction pH and temperature for both soluble and immobilized enzyme were around 8.0 and 45 degrees C, respectively. The free enzyme was stable in the pH range from 5.0 to 11.0, whereas the immobilized enzyme tended to detach from the gel at pH values higher than 9.0. Both forms of enzyme were stable up to 40 degrees C. When used for the optical resolution of N-acetyl-DL-methionine, the immobilized enzyme maintained 90% initial activity after 17 days of continuous operation at 45 degrees C. The process of purification and immobilization of D-aminoacylase described in this report is very effective and easy to scale up.     

Immobilization of glucansucrase for the production of gluco-oligosaccharides from Leuconostoc mesenteroides

Glucansucrase from Leuconostoc mesenteroides was immobilized in 1?% (w/v) with sodium alginate to produce oligosaccharides. Glucansucrase gave three activity bands of approx. 240, 178, and 165?kDa after periodic acid-Schiff staining with sucrose. The immobilized enzyme had 40?% activity after ten batch reactions at 30?°C and 75?% activity after a month of storage at 4?°C, which is six times more stable than the free enzyme. Immobilized enzyme was more stable at lower (3.5?4.5) and higher (6.5?7.0) pH ranges and higher temperatures (35?40?°C) compared with the free enzyme. Immobilized and free glucansucrase were employed in the acceptor reaction with maltose and each produced gluco-oligosaccharide ranging from trisaccharides to homologous pentasaccharides.     

Stability analysis ofBacillus stearothermopilus L1 lipase fused with a cellulose-binding domain

This study was designed to investigate the stability of a lipase fused with a cellulose-binding domain (CBD) to cellulase. The fusion protein was derived from a gene cluster of a CBD fragment of a cellulase gene inTrichoderma hazianum and a lipase gene inBacillus stearothermophilus L1. Due to the CBD, this lipase can be immobilized to a cellulose material. Factors affecting the lipase stability were divided into the reaction-independent factors (RIF), and the reaction-dependent factors (RDF). RIF includes the reaction conditions such as pH and temperature, whereas substrate limitation and product inhibition are examples of RDF. As pH 10 and 50°C were found to be optimum reaction conditions for oil hydrolysis by this lipase, the stability of the free and the immobilized lipase was studied under these conditions. Avicel (microcrystal-line cellulose) was used as a support for lipase immobilization. The effects of both RIF and RDF on the enzyme activity were less for the immobilized lipase than for the free lipase. Due to the irreversible binding of CBD to Avicel and the high stability of the immobilized lipase, the enzyme activity after five times of use was over 70% of the initial activity.