Enzyme and Microbial Technology最新文献

Lignin depolymerization, which enables the breakdown of a complex and heterogeneous aromatic polymer into relatively uniform derivatives, serves as a critical process in valorization of lignin. Enzymatic lignin depolymerization has become a promising biological strategy to overcome the heterogeneity of lignin, due to its mild reaction conditions and high specificity. However, the low solubility of lignin compounds in aqueous environments prevents efficient lignin depolymerization by lignin-degrading enzymes. The employment of biocompatible ionic liquids (ILs) and deep eutectic solvents (DESs) in lignin fractionation has created a promising pathway to enzymatically depolymerize lignin within these green solvents to increase lignin solubility. In this review, recent research progress on enzymatic lignin depolymerization, particularly in a consolidated process involving ILs/DESs is summarized. In addition, the interactions between lignin-degrading enzymes and solvent systems are explored, and potential protein engineering methodology to improve the performance of lignin-degrading enzymes is discussed. Consolidation of enzymatic lignin depolymerization and biocompatible ILs/DESs paves a sustainable, efficient, and synergistic way to convert lignin into value-added products.

Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl₃, Na₂S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in S. blattae, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H₂-oxidizing C. necator H16, a novel host for the expression of RDases.

The split-GFP system is a versatile tool with numerous applications, but it has been underutilized for the labeling of heterologous surface-displayed proteins. By inserting the 16 amino acid sequence of the GFP11-tag between a protein of interest and an autotransporter protein, it is possible to present a protein at the outer membrane of gram-negative bacteria and to fluorescently label it by complementation with externally added GFP1–10. The labeled cells could be clearly discerned from cells without the protein of interest using flow cytometry and the insertion of the GFP11-tag caused no significant alteration of the catalytic activity for the tested model enzyme CsBglA. Furthermore, the amount of the protein of interest on the cells could be quantified by comparing the green fluorescence resulting from the complementation to that of standards with known concentrations. This allows a precise characterization of whole-cell biocatalysts, which is difficult with existing methods. The split-GFP complementation approach was shown to be specific, in a similar manner as commercial antibodies. It is cost-efficient, minimizes the possibility of adverse effects on protein expression or solubility, and can be performed at high throughput.

Cyanide is widely utilized in the extraction of precious metal extraction even though it has been deemed as the most toxic compound. Fusarium oxysporum has been shown to degrade cyanide through the activity of the Nitrilase enzyme. In this study, the coding sequence of nitrilase gene from F. oxysporum genomic DNA was optimized for cloning and expression in E. coli. The pUC57 containing synthetic optimized nitrilase gene was transferred into E. coli DH5α strain. This nitrilase gene was sub-cloned into pET26b (+) expression vector containing an in-built His-tag at the C-terminal end to facilitate its purification. The recombinant plasmid, pETAM1, was confirmed by PCR, digestion pattern, and sequencing. The recombinant protein was overproduced in E. coli BL21 (DE3). The results of the SDS-PAGE pattern and Western blot analysis confirmed the expression of the expected recombinant protein. For expression optimization of Nitrilase protein, M16 orthogonal experimental design of the Taguchi method was used. The effect of induction time, temperature and IPTG concentration were examined using four levels for each factors. Estimation of the amount of the expressed protein was calculated via densitometry on SDS-PAGE. The enzyme activity and expression in E. coli proved to be successful since there was ammonia production when potassium cyanide and acrylonitrile were used as substrates while the highest enzyme activity of 88% was expressed at 30 °C. The K_m and V_m values of the expressed Nitrilase enzyme were determined to be 0.68 mM and 0.48 mM/min respectively.

Indigo, an economically important dye, could be biosynthesized from indole by catalysis of the styrene monooxygenase StyAB. To enhance indigo biosynthesis, the styAB gene and its transcription regulator gene styS/styR in styrene catabolism were cloned from Pseudomonas putida and coexpressed in Escherichia coli. The presence of the intact regulator gene styS/styR dramatically increased the transcriptional levels of styA and styB by approximately 120-fold in the recombinant strain SRAB2 with coexpression of styS/styR and styAB compared to the control strain ABST with solo expression of styAB. A yield of 67.6 mg/L indigo was detected in strain SRAB2 after 24 h of fermentation with 120 μg/mL indole, which was approximately 14-fold higher than that in the control strain ABST. The maximum yield of indigo was produced from 160 μg/mL indole in fermentation of strain SRAB2. However, the addition of styrene to the media significantly inhibited the transcription of styA and styB and consequent indigo biosynthesis in recombinant E. coli strains. Furthermore, the substitution of indole with tryptophan as the fermentation substrate remarkably boosted indigo production, and the maximal yield of 565.6 mg/L was detected in strain SRAB2 in fermentation with 1.2 mg/mL tryptophan. The results revealed that the regulation of styAB transcription by the two-component regulator StyS/StyR in styrene catabolism in P. putida was effective in E. coli, which provided a new strategy for the development of engineered E. coli strains with the capacity for highly efficient indigo production.

Sepsis is a serious disease caused by an impaired host immune response to infection, resulting in organ dysfunction, tissue damage and is responsible for high in-hospital mortality (approximately 20%). Recently, WHO documented sepsis as a global health priority. Nevertheless, there is still no effective and specific therapy for clinically detecting sepsis. Nanomaterial-based approaches have appeared as promising tools for identifying bacterial infections. In this review, recent biosensors are introduced and summarized as nanomaterial-based platforms for sepsis management and severe complications. Biosensors can be used as tools for the diagnosis and treatment of sepsis and as nanocarriers for drug delivery. In general, diagnostic methods for sepsis-associated bacteria, biosensors developed for this purpose are presented in detail, and their strengths and weaknesses are discussed. In other words, readers of this article will gain a comprehensive understanding of biosensors and their applications in sepsis management.

Xanthine is derived from hypoxanthine by xanthine oxidase (XOD), a flavoprotein containing molybdenum and non-haem iron, sulfur and from guanine by guanine deaminase enzyme. Xanthine is oxidized into uric acid by XOD. Xanthine is used as an indicator of fish freshness, based on the reactions in which ATP is degraded into xanthine and its quantity increases with time of fish death. Fresh fish meat is required in food industry for making high quality items. The determination of xanthine in biological fluids is also used in diagnosing and curing many diseases like renal failure, gout, xanthinuria, hyperuricemia. Various methods are available for detection of xanthine but most of them are complicated, time consuming less sensitive & specific and require expensive instrumental setup and trained person to operate. Enzyme based biosensors and non enzymic sensors overcome these disadvantages, as these are simple, rapid, specific, sensitive and easy to operate. Present review describes xanthine biosensors, which work optimally between pH 3.5–9.0, temperature 25 °C-65 °C, xanthine concentration ranging from 0.001–50 × 10⁴ µM. These biosensors have also been used to measure xanthine concentration in beverages, urine and serum samples. Various modified electrodes have been discussed for the detection of xanthine using both enzymatic and non-enzymatic approaches in the present review.

γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene (gadz11) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability, a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5′-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration, cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.

The enzymes of the mevalonate pathway need to be improved to achieve high yields of isoprenoids in the yeast Saccharomyces cerevisiae. The red yeast Rhodosporidium toruloides produces high levels of carotenoids and may have evolved to carry a naturally high flux of isoprenoids. Enzymes from such yeasts are likely to be promising candidates for improvement. Towards this end, we have systematically investigated the various enzymes of the mevalonate pathway of R. toruloides and custom synthesized, expressed, and evaluated six key enzymes in S. cerevisiae. The two nodal enzymes geranyl pyrophosphate synthase (RtGGPPS) and truncated HMG-CoA reductase (RttHMG) of R. toruloides showed a significant advantage to the cells for isoprenoid production as seen by a visual carotenoid screen. These two were analyzed further, and attempts were also made at further improvement. RtGGPPS was confirmed to be superior to the S. cerevisiae enzyme, as seen from in vitro activity determinations and in vivo production of the heterologous diterpenoid sclareol. Four mutants were created through rational mutagenesis but were unable to improve the activity further. In the case of RttHMG, functional evaluation of the enzyme revealed that it was very unstable despite functioning very well in S. cerevisiae. We succeeded in stabilizing the enzyme through mutation of a conserved serine in the catalytic region, which did not alter the enzyme activity per se. In vivo evaluation of the mutant revealed that it could enable better sclareol yields. Therefore, these two enzymes from the red yeast are excellent candidates for heterologous isoprenoid production.