重组角质酶的发酵制备及其对涤纶纤维的表面改性

对大肠杆菌表达嗜热子囊菌Thermobifida fusca角质酶的摇瓶诱导条件及3 L发酵罐扩大培养进行了研究,并探讨了角质酶对涤纶纤维的改性作用。结果表明,在摇瓶培养中,采用工业级TB培养基,用2 g/L乳糖诱导,菌体培养至对数生长前期添加0.5％甘氨酸,角质酶产量可达到128 U/mL。在3 L发酵罐扩大培养中,补料培养生物量 (OD600) 最大达到35,角质酶酶活最高达506 U/mL,是迄今国内外报道细菌来源角质酶的最高水平。紫外分光光度法分析初步表明涤纶纤维经角质酶水解产生了对苯二甲酸类物质     

评论：塑料结合模块促进聚对苯二甲酸乙二醇酯的酶法降解

塑料的大量生产和无节制的使用已造成严重的环境污染。为了减少塑料废物对环境的影响,近年来塑料酶法降解已成为国内外研究者关注的热点。例如,通过蛋白质工程策略提高塑料降解酶催化活性和热稳定性,进一步提高酶法降解的效率。另外,通过融合酶策略将塑料结合模块与塑料降解酶融合,也可以促进塑料降解。近期发表在期刊Chem Catalysis的一项研究表明,采用碳水化合物结合模块融合策略可以在低浓度(<10 wt%)的底物聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]中提高塑料降解酶的活性。但是在高浓度底物(10 wt%−20 wt%)中,该策略无法提高PET的酶法降解。该项研究对于采用塑料结合模块促进酶法降解塑料具有重要的指导意义。     

Hydrolysis of cyclic poly(ethylene terephthalate) trimers by a carboxylesterase from Thermobifida fusca KW3

We have identified a carboxylesterase produced in liquid cultures of the thermophilic actinomycete Thermobifida fusca KW3 that were supplemented with poly(ethylene terephthalate) fibers. The enzyme hydrolyzed highly hydrophobic, synthetic cyclic poly(ethylene terephthalate) trimers with an optimal activity at 60°C and a pH of 6. V _max and K _m values for the hydrolysis were 9.3 μmol⁻¹ min⁻¹ mg⁻¹ and 0.5 mM, respectively. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. The enzyme retained 37% of its activity after 96 h of incubation at 50°C and a pH of 8. Enzyme inhibition studies and analysis of substitution mutants of the carboxylesterase revealed the typical catalytic mechanism of a serine hydrolase with a catalytic triad composed of serine, glutamic acid, and histidine.     

Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules

A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.     

Upcycling of poly(ethylene terephthalate) to produce high-value bio-products

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来源于糖丝菌双(2-羟乙基)对苯二甲酸酯水解酶的酶学性质表征及降解特性分析

聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]降解酶的发掘是国内外研究的热点。双(2-羟乙基)对苯二甲酸酯[bis-(2-hydroxyethyl)terephthalic acid,BHET]是PET降解过程的一种中间化合物,会与PET竞争酶的底物结合位点,从而抑制PET进一步降解。因此,探寻新型BHET降解酶,对进一步提高PET的降解效率具有促进作用。本研究通过基因挖掘发现了一种来源于浅黄糖丝菌(Saccharothrix luteola)参与PET降解过程的水解酶基因sle(ID:CP064192.1,5085270–5086049),其编码的蛋白质可以将BHET水解为单(2-羟乙基)对苯二甲酸酯[mono-(2-hydroxyethyl)terephthalate,MHET]和对苯二甲酸(terephthalic acid,TPA)。将BHET水解酶(Sle)通过重组质粒在大肠杆菌(Escherichia coli)中异源表达,结果表明,在异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导终浓度为0.4 mmol/L,诱导时长为12 h,诱导温度为20℃时蛋白的表达量最高。通过镍亲和层析、阴离子交换层析和凝胶过滤层析3步分离纯化,获得了高纯度的Sle重组蛋白;同时对其酶学性质进行了表征,Sle最适温度和pH分别为35℃和8.0,在25–35℃和pH 7.0–9.0区间内能保持80%以上的残余酶活,且金属离子Co^(2+)能提高酶活力;进一步通过同源序列及Sle复合物结构分析得知,该酶属于二烯酸内酯水解酶(dienelactone hydrolase,DLH)家族,具备该家族典型的催化三联体,预测其催化位点分别为S129、D175和H207,并初步分析了其催化机理。最后,利用高效液相色谱法(high performance liquid chromatography,HPLC)鉴定了该酶能够特异性降解BHET生成MHET和TPA,属于BHET降解酶。本研究为生物酶法高效降解PET塑料提供了新的酶资源。     

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL)

BACKGROUND: Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. METHODS: A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. RESULTS: We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. CONCLUSIONS: PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.     

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.     

Micropatterns of cell adhesive proteins with poly(ethylene oxide)-block-Poly(4-vinylpyridine) diblock copolymer

Control of cell shape and behavior through the micropattern technique by spatial immobilization of adhesive proteins on a surface has provided novel insights in several aspects of cell biology, such as tissue morphogenesis, cell growth and cell differentiation, and apoptosis. In this work, we present the use of poly(ethylene oxide-block-poly(4-vinylpyridine) (PEO-b-P4VP) as a non-adhesive background to construct micropatterns of cell adhesive proteins. In the method presented, PEO-b-P4VP is used for its antifouling properties and at the same time, as a photosensitive material to define the micropatterns. The irradiation of PEO-b-P4VP with a short wavelength UV light through photolithographic mask, causes the polymer to crosslink and immobilize in the areas exposed. In the areas non-exposed the polymer can be removed. These areas can be subsequent back filled with the adhesive protein of interest to produce the final micropatterned cell chips.     

Poly(ethylene glycol) cross-linked hemoglobin with antioxidant enzymes protects pancreatic islets from hypoxic and free radical stress and extends islet functionality

The objective of this study was to investigate the efficiency of multifunctional poly(ethylene glycol)-based hemoglobin conjugates crosslinked with antioxidant enzymes for their ability to protect an oxygen carrier (hemoglobin) and insulin secreting islets from the combination of hypoxic and free radical stress under simulated transplantation conditions. In this study, RINm5F cells and isolated pancreatic islets were challenged with oxidants (H(2)O(2) or xanthine and xanthine oxidase) and incubated with conjugates (hemoglobin-hemoglobin or superoxide dismutase-catalase-hemoglobin) in normoxia (21% oxygen) or hypoxia (6% or 1% oxygen). Hemoglobin protection, intracellular free radical activity and cell viability in RINm5F cells measured by methemoglobin, dichlorofluorescein-diacetate, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, respectively, showed that cells were better protected by conjugates containing antioxidant enzymes. Insulin secretion from islets and qualitative confocal evaluation of viability showed beta cells were protected by conjugates containing antioxidant enzymes when exposed to induced stress. Our study suggested that antioxidant enzymes play a significant role in hemoglobin protection and thus extended cell protection.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Enhancement of the adhesion of fibroblasts by peptide containing an Arg-Gly-Asp sequence with poly(ethylene glycol) into a thermo-reversible hydrogel as a synthetic extracellular matrix

In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.     

Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2_IIA3_IIB_IIA1_II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.