查看更多>>摘要：In this study, a novel kind of Ni-NTA modified monodispersed SiO2 nanoflowers (Ni-NTA@SiO2 nanoflowers) were successfully synthesized. The obtained Ni-NTA@SiO2 nanoflowers were used to specifically adsorb and purify His-tagged old yellow enzyme (OYE1) and glucose dehydrogenase (GDH), which allows access to optically pure (3 S)-3-methyl-cyclohexanone through asymmetric hydrogenation reaction, and forms a cofactor regeneration system. The protein loading amount on Ni-NTA@SiO2 nanoflowers was 40.17 mg/g support and the activity recoveries of OYE1 and GDH were 81.53% and 79.68%, respectively. The effects of pH and temperature on the activity of free and co-immobilized enzymes were investigated, and the stability as well as reusability were also measured. Compared to free enzymes, the co-immobilized enzymes showed higher thermal and storage stability. The co-immobilized enzymes were applied to asymmetric reduction of C--C bonds for the synthesis of a chiral center with excellent enantioselectivity (ee > 99%), and the conversion was 46.02% after 7 cycles. This work introduced a one-pot multi-enzyme purification and co-immobilization strategy to construct efficient cofactor regeneration system with high activity and stability.
查看更多>>摘要：Carotenoids, an important kind of natural pigments with great potential commercial value, have been widely used in nutrition and health care, cosmetics and aquaculture industries. Schizochytrium sp. is a potential cell factory for lipid nutrition chemicals production including docosahexaenoic acid and carotenoids. The purpose of this study is to mine and identify the carotenoid biosynthesis genes in Schizochytrium sp. Firstly, based on the genomic information of Schizochytrium sp., we obtained the gene sequences of a trifunctional enzyme (CrtIBY), carotene hydroxylase (CrtZ) and carotene ketolase (CrtO) in carotenoids biosynthesis pathway by bioinformatics analysis. Subsequently, using the lycopene-producing E. coli as the host, 22.77 ug/L of 8-carotene and 44.31 ug/L of zeaxanthin were synthesized by overexpression of CrtIBY and further co-expression with CrtZ from Schizochytrium sp. After that, 54.78 ug/L of astaxanthin was synthesized using hydroxylase and ketonase from Haematococcus pluvialis. The key enzymes for carotenoids biosynthesis identified in this study is of great significance for further understanding the metabolic mechanism in Schizochytrium sp, which could also provide the functional elements and theoretical support for astaxanthin production.
查看更多>>摘要：Flavonoids are a group of valuable compounds with a variety of health benefits. (2 S)-Naringenin is an important flavonoid skeleton, which can be tailored into almost all flavonoids. In this study, the Saccharomyces cerevisiae native precursor pathways were explored and higher-active CHSs from plants rich in flavonoids were screened. The results indicated that overexpressing the native precursor pathways is not an efficient approach to improving (2 S)-naringenin production in our chassis strain. On the other hand, by screening from plants rich in flavonoids, we obtained four CHSs with higher activities than the commonly used PhCHS. Among these CHSs, SjCHS1 increased the (2 S)-naringenin titer by 48.38% in shaking flasks. Finally, we combined the native precursor pathways optimization with the higher-active CHS that screened, and further increased the (2 S)-naringenin titer to 203.49 mg/L from glucose in shaking flasks. The results achieved in this study indicated that plants rich in flavonoids are good sources for higher-active CHS screening, and that the heterologous pathway and chassis precursor flux should be synergistically engineered to achieve higher production.
查看更多>>摘要：In this study, a ligninolytic enzyme-producing strain F5 was isolated and identified as Bacillus thuringiensis, which can efficiently degrade methylene blue (MB) dye. The optimal pH, temperature, rotation speed, NaCl concentration, and inoculum of strain F5 for MB degradation were pH 6.0, 30 ?C, 140 rpm, 10 g/L NaCl, 4% inoculum (v/v), and the strain F5 had salt tolerance, the MB decolorization rate reached 95% after 12 h. The degraded products were characterized by UV-vis, FT-IR, and GC-MS. Based on products analysis, four different intermediates were identified, and a new pathway for the degradation of MB was proposed. The degradation of MB by strain F5 was due to the synergistic effects of laccase (Lac), manganese peroxidase (MnP), lignin peroxidase (LiP), and NADH-DCIP reductase; among them, Lac and MnP were the key enzymes. The phytotoxicity results showed that MB degraded metabolites' toxicity was lower than that of the parent compound, indicating that the strain F5 had a detoxification effect on MB dyes.
查看更多>>摘要：Thermomicrobium roseum DSM 5159 lipase (TrLip) is an enzyme with marked thermostability and excellent solvent resistance. However, TrLip reveals relatively high catalytic efficiency on short-chain substrates but poor activity against mid-long or long-chain fatty acids, which would limit its industrial application. In this study, ancestral sequence reconstruction (ASR), a common engineering tool for the evolutionary history of protein families, was employed to identify the natural evolutionary trends within 5 angstrom around the catalytic center. Two mutation libraries were constructed, one for the catalytic center and the other for the pocket flexibility. A total of 69 mutants were expressed and purified in the Escherichia coli expression system to determine the kinetic parameters, and W219G could significantly enhance the catalytic efficiency against substrates with 12-, 16- and 18carbon side chains. In addition, the double mutant W219G/F265M could further catalyze the breakdown of the above three substrates up to 6.34-, 4.21- and 4.86-folds compared to the wild-type TrLip, while the initial pH and thermostability were maintained. Through bioinformatics analysis, the significantly enhanced catalytic efficiency against longer-side chain substrates should be associated with the reduction of steric hindrance. With the outstanding stability and the promoted activity, TrLip should be of great potential in chemical and food industry.
查看更多>>摘要：Mycobacterium smegmatis strain mc(2) 155 is a fast-growing and non-pathogenic mycobacteria and widely used in genetic studies of mycobacteria. It has been shown that this species of mycobacterium can transfer its genomic DNA fragments to other species of mycobacteria during the conjugation process. Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal) glycan epitope is a highly immunogenic epitope exerted by the enzyme alpha 1-3-galactosyltransferase (alpha 1,3GT) in mammalian cells on the glycan skeleton. However, the enzyme is inactive in humans, primates and Old World monkeys as a result of evolutionary mutations. The robust immunogenicity induced by the epitope in human, has attracted much attention to apply the epitope in vaccine research. In this study we proved successful transfer of desired expression cassettes from fast-growing Mycobacterium smegmatis mc(2 )155, to the slow-growing pathogen Mycobacterium tuberculosis H37Rv. We designed gene cassettes encoding the alpha 1,3GT enzyme under control the potent G13 promoter and the cassette containing hygromycin resistance gene under a Mtb specific promoter, Ptpa in the vector pMV306DIG13 +FflucRT (harboring attP site). The resulting construct was electroporated into mc(2) 155 strain in combination with pBS-int containing the gene encoding Mycobacteriophage L5 integrase to integrate pMV306DIG13 +FflucRT-cassettes into mc(2) 155 genome. Following the integration, the recombinant clones were placed in vicinity to the Mycobacterium tuberculosis H37Rv strain to establish conjugation. Conjugated recombinant clones were selected on the medium containing the hygromycin B and transfer of the desired cassettes to Mycobacterium tuberculosis was confirmed. The enzyme alpha 1,3GT in transconjugants were also investigated.
查看更多>>摘要：Phytase belongs to orthophosphate monoester hydrolase, which can catalyze the gradual hydrolysis of phytic acid to inositol phosphate. It can be added to animal feed to reduce the anti-nutritional factor of phytic acid in feed. The thermostability and specific activity of phytases are two key factors determining their potential applications. In this study, a highly active 233-aa phytase gene (LpPHY233) from Lactobacillus plantarum was cloned and expressed in Escherichia coli (E. coli), achieving 800 times higher activity than that expressed in L. plantarum. Next, the temperature characteristic and catalytic performance of LpPHY233 was improved by disulfide bond engineering and C-terminal truncation, respectively. Surprisingly, the specific activity of the C-terminal truncated mutant LpPHY200 was about 5.6 times higher than that of LpPHY233, and the optimal temperature for the mutant LpPHY233S58C/K61C introduced disulfide bond was 15 C higher than that of LpPHY233. Moreover, these phytase mutants displayed excellent pH property and kinetic parameters, and have great application prospect in feed additives field. The molecular basis for its catalytic performance was preliminarily explained by in silico design methods. Our results provided a solid theoretical foundation for further molecular modification and industrial application of phytases.
查看更多>>摘要：Glycoside hydrolase (GH) family 10 and 11 xylanases are inhibited by many xylanase inhibitor proteins (XIPs). We recombinantly expressed the Oryza sativa xylanase inhibitor protein (OsXIP) in Pichia pastoris GS115, with a molecular mass of 47.0 kDa. Family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the mutant T33I (DS199) were inhibited by the recombinant OsXIP (rePOsXIP) through competive inhibition, with corresponding inhibition constants (K-i) of 54.09 and 12.16 nM. After incubation with rePOsXIP (70 nM) at 40 ?C for 40 min, inhibitory rates of reBaxA and DS199 (0.2 U) were 23.7% and 76.7%, respectively. Xylooligosaccharides with low concentration were released from beechwood xylan by reBaxA and DS199 in the presence of reOsXIP. Intrinsic fluorescences of reBaxA and DS199 were statically quenched by rePOsXIP in a concentration-dependent manner. Molecular dynamics (MD) simulations and conformational analysis of OsXIP-BaxA and OsXIP-DS199 revealed that the long loop (L alpha(4)beta(5)) of OsXIP inserted into the catalytic grooves of BaxA and DS199. The DS199 enhanced the binding affinity to OsXIP, causing conformational alterations on protein-protein interface residues, thereby forming more hydrogen bonds and van der Waals forces. MM/GBSA analysis revealed that the binding free energy (delta G(bind)) of OsXIP-DS199 was enhanced by 2.08 kcal/mol compared to that of OsXIP-BaxA. The OsXIP binding induced a conformational changes among residues in the cord and thumb regions of BaxA and DS199. In particular, the T(111)RYNAP(116) residues in the thumb region of DS199 was maintained close to OsXIP by specific bonds. Additional MD simulations revealed that Y113A or T93A mutation of BaxA suppressed the binding affinity by diminishing interface associations of OsXIP-BaxA. This study partially elucidats the molecular basis of inhibitory mechanism and structure-function relationships of GH11 xylanases. Our findings inform rational designs of mutant xylanases with higher resistance to inhibitor proteins.
查看更多>>摘要：The composition of cellulosomal carbohydrate-active enzymes (CAZymes) secreted from a cellulolytic bacterium Clostridium thermocellum varies depending on the cellulosic substrate used during cultivation. C. thermocellum detects the polysaccharides in cellulosic material via anti-sigma factors and expresses the appropriate CAZyme gene via alternative sigma factors, SigIs. Previous studies on the regulation of CAZyme gene expression via SigIs in C. thermocellum have been conducted in vitro or in a heterologous host, because of the limited genetic tools available for C. thermocellum. To characterize the in vivo function of SigIs, in the present study, we established a sigI7 gene expression strain of C. thermocellum. Transcriptome analysis of this strain revealed that SigI7 induced the expression of cellulosomal CAZyme genes and cellulosomal scaffold genes. However, there was a decrease in the degradation ability of the exoproteome from the sigI7 expression strain; the product of the downregulated gene, Clo1313_1002, rescued the activity of the C. thermocellum exoproteome from the sigI7 expression strain. In this study, we demonstrate the in vivo function of SigI7 and discuss the CAZymes that are important for cellulosic biomass degradation by C. thermocellum.
查看更多>>摘要：Biosensors have emerged as a potential tool for selective and sensitive detection. Biomolecules like enzymes, deoxyribonucleic acid (DNA), and antibodies are used as recognition sites in biosensors due to their high selectivity, sensitivity, and signal-to-noise ratio. Nowadays, biosensors are used to detect many vital biomolecules such as glucose, urea, and cholesterol. Exploiting enzymes for the selective detection of target molecules are very good but may not be easily accomplished as enzymes often need to be immobilized onto support materials, which requests either modifying the surface or using appropriate linkers to synthesize biosensors. Currently, many biosensors' signal strength and stability still are low, which encourages the fabrication of functionalized nanomaterials/nanoparticles to enhance electrochemical performance. The review covers current progress in modifying enzyme immobilized biosensors to detect biologically essential molecules (glucose, urea, cholesterol). A discussion of the different types of enzyme immobilization is also provided. This review may be helpful for researchers to build rational and novel biosensors for efficient enzyme immobilization and sensing applications.
NSTL
Elsevier