A comparison of entrapped and covalently bonded laccase: Study of its leakage,reusability, and the catalytic efficiency in TEMPO-mediated glycerol oxidation

This article presents the comparison for reusability and leakage between entrapped and covalently bonded laccase and their performances towards the selective oxidation of glycerol. The reusability of immobilized laccase enzyme was studied by reacting a batch of immobilized laccase with ABTS for 15 cycles. The investigation of the leakage of immobilized laccase was carried out by storing the immobilized laccase in acetate buffer solution for 32 days. The data show that the retained enzyme activities of entrapped and covalently bonded enzyme after being reused for eight cycles were well above 60% and the leakages after storing for a month in the acetate buffer at 4?°C were well below 15%. The entrapped laccase coupled with TEMPO was found to perform better and gave a two-fold higher yield of glyceraldehyde and glyceric acid in the selective oxidation of glycerol compared to covalently bonded laccase. Hence, physical entrapment of laccase would be a suitable immobilization method in the laccase-mediated selective oxidation of glycerol.     

Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers

Extracellular laccases produced by three different wood-rotting fungi, Cerrena unicolor, Heterobasidion annosum and Trametes versicolor, were immobilized via covalent bonds formation on DEAE-Granocel 500, CM-Granocel 500, and acrylic carriers. Out of the tested carriers, only the DEAE-Granocel 500, which was activated by divinyl sulphone appeared to be a suitable matrix for the expression of enzymic activity. Only one laccase of all the tested enzymes produced by C. unicolor showed the best binding to the carrier and a satisfactory enzymic activity. The immobilized laccase exhibited the highest enzymic activity at pH 5.2 and it was more resistant to thermal denaturation than the native enzyme. At 90 °C, it retained 75% activity compared to the free enzyme. It was also more stable during storage at 4 °C: after 4 months the immobilized laccase retained 98% of initial activity. Immobilized C. unicolor laccase was active in 10–60% concentration of methanol, acetone, isopropanol or acetonitrile. The best enzymic activity was observed in 20% solution of acetonitrile in buffer.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

柱状田头菇生长发育中9种胞外酶活性的测定

柱状田头菇Agrocybe aegerita作为一种新开发的食用菌,越来越受到消费者的喜爱和研究者的关注,但在其栽培生产中遇到的产量相对较低的问题,一直未能得到很好的解决,致使柱状田头菇的栽培难以得到大规模的推广。本研究对不同来源的柱状田头菇在其6个不同生长发育阶段中,9种与培养料中主要组分分解相关的胞外酶活性的变化进行了测定,结果表明：柱状田头菇属褐腐菌,对非木质纤维素的利用能力最强,对纤维素的利用能力较强,对木质素的利用能力较差,但柱状田头菇具有漆酶活性。     

含双酚A废水植物修复技术研究进展

双酚A作为一种重要的生产原料;近几十年;在环境中的含量剧增。双酚A是一种内分泌干扰激素类物质;环境激素类物质能够模拟、强化和抑制激素作用;在某些情况下;引发组织或者器官增生和肿瘤。植物修复技术是近年来发展起来的一种环境污染修复技术;是生物修复领域的一个热点。在阅读大量文献的基础上;对修复双酚A植物的筛选进行了概括;从植物糖基化修饰效应、根系分泌酶催化降解、植物根际与微生物联合代谢作用三个方面阐述植物修复双酚A的主要机理;对国内外近年来植物修复技术在含双酚A废水修复中的应用、研究成果进行了综述;并对今后的该领域的研究进行了展望;旨在为双酚A废水的植物修复技术提供参考和借鉴。     

Distribution of Cysteine Desulfhydrase in Microorganisms

The distribution of cysteine desulfhydrase activity in microorganisms was studied with intact cells. The enzyme activity was found mainly in strains belonging to Enterobacteriaceae, especially to genus Aerobacter (Enterobacter). Aerobacter cloacae IFO 12009 showed markedly high activity.

l-Cysteine was essential as an inducer of the enzyme formation, of which 0.2% in the medium is appropriate.

Intact cells of bacteria containing high cysteine desulfhydrase activity, prepared from broth cultured for 19hr, catalyzed the synthesis of l-cysteine from pyruvate, ammonia and hydrogen sulfide.     

Determination of bisphenol A using a flow injection inhibitory chemiluminescence method.

A new flow injection chemiluminescence (CL) method has been developed for the determination of bisphenol A (BPA), based on the inhibitory effect of BPA on the chemiluminescence reaction between luminol and potassium hexacyanoferrate. Under optimum conditions, the decrease in CL emission intensity was linear with BPA concentration in the range 8.0 x 10(-7)-1.2 x 10(-5) mol/L, and the detection limit was 3.1 x 10(-7) mol/L. The relative standard deviation (RSD) of 11 replicate measurements was 2.6% for 2.0 x 10(-6) mol/L BPA (n = 11). The sampling frequency was calculated to be ca. 120/h. This method has been successfully used to determine the content of BPA in aqueous solution of polycarbonate materials. A brief discussion on the possible chemiluminescence reaction mechanism is presented.     

葡萄糖淀粉酶在高压静电纺PEI/PVA纳米纤维膜上的离子吸附固定

旨在应用离子结合法将葡萄糖淀粉酶固定在PEI/PVA纳米纤维膜上并对其理化性质进行研究。采用高压静电纺丝技术制备聚乙烯亚胺(PEI)/聚乙烯醇(PVA)纳米纤维膜,采用热交联方法使其具备水稳定性后,再利用离子吸附法固定葡萄糖淀粉酶。结果显示,利用红外光谱(FT-IR)表征固定有葡萄糖淀粉酶的PEI/PVA纳米纤维膜,表明葡萄糖淀粉酶可成功固定在静电纺丝形成的PEI/PVA纳米纤维膜表面。通过固定化葡萄糖淀粉酶的酶学性质鉴定,发现固定化葡萄糖淀粉酶的最适反应温度为65℃,比游离的葡萄糖淀粉酶提高了6℃;固定化葡萄糖淀粉酶的适用p H值范围明显变宽;热稳定性和存贮稳定性显著增强且可以重复使用。利用离子吸附法能简便地将蛋白质分子固定于纳米纤维膜上,具有一定的应用前景。     

以伴刀豆球蛋白为介质定向固定化脲酶的研究

将戊二醛将伴刀豆球蛋白(ConA)和壳聚糖载体交联,然后利用ConA与脲酶糖链的特异性结合作用,实现脲酶的定向固定化.定向固定化的最适条件为戊二醛浓度3.5%、ConA浓度1mg/mL、ConA溶液pH值7.0、脲酶浓度0.4mg/mL.定向固定化脲酶的最适pH 5.0～6.0、最适温度77℃,米氏常数Km11.76mmol/L,与游离酶及非定向固定化脲酶比较,定向固定化脲酶的最适pH向酸性范围发生了偏移并有更宽的pH适用范围,最适温度提高,与底物的亲和力较大,且有较好的操作稳定性.     

以伴刀豆球蛋白为介质定向固定化脲酶的研究

将戊二醛将伴刀豆球蛋白(ConA)和壳聚糖载体交联, 然后利用ConA与脲酶糖链的特异性结合作用, 实现脲酶的定向固定化。定向固定化的最适条件为戊二醛浓度3.5%、ConA浓度1 mg/mL、ConA溶液pH值7.0、脲酶浓度 0.4 mg/mL。定向固定化脲酶的最适pH 5.0~6.0、最适温度77°C、米氏常数Km11.76 mmol/L, 与游离酶及非定向固定化脲酶比较, 定向固定化脲酶的最适pH向酸性范围发生了偏移并有更宽的pH适用范围, 最适温度提高, 与底物的亲和力较大, 且有较好的操作稳定性。     

Synthesis of Glycoglycerolipids: 3-O-Mannooligosyl-l,2-di-O-tetradecyl-sn-glycerol

Synthetic routes for the following mannooligosylglycerolipids of biological interest were developed by using regioselectively protected monosaccharide synthons and l,2-di-O-alkyl-sn-glycerol; 3-O-(2-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-l,2-di-O-tetradecyl-sn-glycerol; 3-O-[2-O-(2-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-α-D-mannopyranosyl]-l,2-di-O-tetradecyl-sn-glycerol; 3-O-(6-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-l,2-di-O-tetradecyl-sn-glycerol; and 3-O-(3,6-di-O-α-D-mannopyranosyl-α-D-mannopyranosyl)-1,2-di-α-tetradecyl-sn-glycerol.     

Determination of bisphenol A in red wine using a double vortex–ultrasound-assisted microextraction assay: Role of the interfacial properties

Bisphenol A (BPA) is a synthetic compound broadly used in medical devices as well as in packaging of food and drinks. Recently, BPA toxicity has become of concern to environmental public health. Red wine that is susceptible to BPA contamination is an alcoholic beverage made from yeast fermentation of grapes in the presence of grape skins so as to extract phenolic compounds. The aim of this study was to validate an efficient, low cost, and time-saving method for BPA determination in red-wine beverage. To this end, a rapid and simple microextraction method is here proposed consisting in liquid–liquid separation assisted by a vortex–ultrasound–vortex procedure combined with gas chromatographic analysis (GC-Fid or GC-IT/MS). By means of a comparative study between real red-wine matrix and synthetic hydroalcoholic solutions, different parameters related to the microextraction steps were investigated. The minimal amount of extraction solvent for a given volume of sample was calculated for both the systems. It was demonstrated that for red-wine matrix, the extent of phase separation is strongly affected by some wine constituents and that separation can be tuned by varying the amount of the extraction solvent. This double vortex–ultrasound-assisted method achieved high recovery of BPA and enrichment factor compared with other microextraction methods.     

Fine‐tuning sortase‐mediated immobilization of protein layers on surfaces using sequential deprotection and coupling

Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer‐by‐layer protein attachment. Here, we report a technique for controlling enzyme‐mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase‐cleavable peptide sequence was inserted at the N‐terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A‐mediated protein immobilization on the surface layer‐by‐layer. Efficient, sequential immobilization of a second layer of protein using Sortase A required removal of the N‐terminal protecting group, suggesting the method enables multilayer synthesis using cyclic deprotection and coupling steps. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:824–831, 2017