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Background: Early detection of hepatocellular carcinoma (HCC) is important in order to improve patient prognosis and survival rate. Methylation sequencing combined with neural networks to identify cell-free DNA (cfDNA) carrying aberrant methylation offers an appealing and non-invasive approach for HCC detection. However, some limitations exist in traditional methylation detection technologies and models, which may impede their performance in the read-level detection of HCC. Methods: We developed a low DNA damage and high-fidelity methylation detection method called No End-repair Enzymatic Methyl-seq (NEEM-seq). We further developed a read-level neural detection model called DeepTrace that can better identify HCC-derived sequencing reads through a pre-trained and fine-tuned neural network. After pre-training on 11 million reads from NEEM-seq, DeepTrace was fine-tuned using 1.2 million HCC-derived reads from tumor tissue DNA after noise reduction, and 2.7 million non-tumor reads from non-tumor cfDNA. We validated the model using data from 130 individuals with cfDNA whole-genome NEEM-seq at around 1.6X depth. Results: NEEM-seq overcomes the drawbacks of traditional enzymatic methylation sequencing methods by avoiding the introduction of unmethylation errors in cfDNA. DeepTrace outperformed other models in identifying HCC-derived reads and detecting HCC individuals. Based on the whole-genome NEEM-seq data of cfDNA, our model showed high accuracy of 96.2%, sensitivity of 93.6%, and specificity of 98.5% in the validation cohort consisting of 62 HCC patients, 48 liver disease patients, and 20 healthy individuals. In the early stage of HCC (BCLC 0/A and TNM I), the sensitivity of DeepTrace was 89.6 and 89.5% respectively, outperforming Alpha Fetoprotein (AFP) which showed much lower sensitivity in both BCLC 0/A (50.5%) and TNM I (44.7%). Conclusions: By combining high-fidelity methylation data from NEEM-seq with the DeepTrace model, our method has great potential for HCC early detection with high sensitivity and specificity, making it potentially suitable for clinical applications. DeepTrace: https://github.com/Bamrock/DeepTrace [ABSTRACT FROM AUTHOR]

Malto-oligosaccharides (MOSs) from starch conversion is advantageous for food and pharmaceutical applications. In this study, an efficient malto-oligosaccharide-forming α-amylase AmyCf was identified from myxobacter Cystobacter sp. strain CF23. AmyCf is composed of 417 amino acids with N-terminal 41 amino acids as the signal peptide, and conserved glycoside hydrolase family 13 (GH13) catalytic module and predicted C-terminal domain with β-sheet structure are also identified. Phylogenetic and functional analysis demonstrated that AmyCf is a novel member of GH13_6 subfamily. The special activity of AmyCf toward soluble starch and raw wheat starch is 9249 U/mg and 11 U/mg, respectively. AmyCf has broad substrate specificity toward different types of starches without requiring Ca2+. Under ideal circumstances of 60 °C and pH 7.0, AmyCf hydrolyzes gelatinized starch into maltose and maltotriose and maltotetraose as the main hydrolytic products with more than 80% purity, while maltose and maltotriose are mainly produced from the hydrolysis of raw wheat starch with more than 95% purity. The potential applicability of AmyCf in starch processing is highlighted by its capacity to convert gelatinized starch and raw starch granules into MOSs. This enzymatic conversion technique shows promise for the low-temperature enzymatic conversion of raw starch. [ABSTRACT FROM AUTHOR]

Methylation sequencing is a promising approach to infer the tissue of origin of cell-free DNA (cfDNA). In this study, a single- and a double-stranded library preparation approach were evaluated with respect to their technical biases when applied on cfDNA from plasma and urine. Additionally, tissue of origin (TOO) proportions were evaluated using two deconvolution methods. Sequencing cfDNA from urine using the double-stranded method resulted in a substantial within-read methylation bias and a lower global methylation (56.0% vs. 75.8%, p ≤ 0.0001) compared to plasma cfDNA, both of which were not observed with the single-stranded approach. Individual CpG site-based TOO deconvolution resulted in a significantly increased proportion of undetermined TOO with the double-stranded method (urine: 32.3% vs. 1.9%; plasma: 5.9% vs. 0.04%; p ≤ 0.0001), but no major differences in proportions of individual cell types. In contrast, fragment-level deconvolution led to multiple cell types, with significantly different TOO proportions between the two methods. This study thus outlines potential limitations of double-stranded library preparation for methylation analysis of cfDNA especially for urinary cfDNA. While the double-stranded method allows jagged end analysis in addition to TOO analysis, it leads to significant methylation bias in urinary cfDNA, which single-stranded methods can overcome. [ABSTRACT FROM AUTHOR]

Objective: The opportunities for sequencing-based methylome analysis of clinical samples are increasing. To reduce its cost and the amount of genomic DNA required for library preparation, we aimed to establish a capture methyl-seq protocol, which adopts pre-pooling of multiple libraries before hybridization capture and TET2/APOBEC-mediated conversion of unmethylated cytosine to thymine. Results: We compared a publicly available dataset generated by the standard Agilent protocol of SureSelect XT Human Methyl-Seq Kit and our dataset obtained by our modified protocol, EMCap, that adopted sample pre-pooling and enzymatic conversion. We confirmed that the quality of DNA methylation data was comparable between the two datasets. As our protocol, EMCap, is more cost-effective and reduces the amount of input genomic DNA, it would serve as a better choice for clinical methylome sequencing. [ABSTRACT FROM AUTHOR]

In traditional oriental medicine, carrots (Daucus carota L.) are considered effective medicinal herbs; however, the use of D. carota leaves (DCL) as therapeutic agents has not been explored in depth. Therefore, we aimed to demonstrate the value of DCL, generally treated as waste while developing plants for wide industrial availability. Six flavone glycosides were isolated and identified from DCL, and their constituents were identified and quantitated using an NMR and HPLC/UV method, which was optimized and validated. The structure of chrysoeriol-7-rutinoside from DCL was elucidated for the first time. The method exhibited adequate relative standard deviation (<1.89%) and recovery (94.89–105.97%). The deglycosylation of DCL flavone glycosides by Viscozyme L and Pectinex was assessed. Upon converting the reaction contents to percentages, the luteolin, apigenin, and chrysoeriol groups showed values of 85.8, 33.1, and 88.7%, respectively. The enzyme-treated DCL had a higher inhibitory effect on TNF-α and IL-2 expression than that of the carrot roots or carrot leaves without enzyme treatments. These results highlight the importance of carrot leaves and could be used as baseline standardization data for commercial development. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Xi'an Polytechnic University is the property of Editorial Department of Journal of Xi'an Polytechnic University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Hydrothermal pretreatment (HTP) has long been considered as an efficient and green treatment process on lignocellulosic biomass for bioconversion. However, the variations of cellulose supramolecular structures during HTP as well as their effects on subsequent enzymatic conversion are less understood. In this work, bamboo holocellulose with well-connected cellulose and hemicelluloses polysaccharides were hydrothermally treated under various temperatures. Chemical, morphological, and crystal structural determinations were performed systematically by a series of advanced characterizations. Xylan was degraded to xylooligosaccharides in the hydrolyzates accompanied by the reduced degree of polymerization for cellulose. Cellulose crystallites were found to swell anisotropically, despite the limited decrystallization by HTP. Hydrogen bond linkages between cellulose molecular chains were weakened due to above chemical and crystal variations, which therefore swelled, loosened, and separated the condensed cellulose microfibrils. Samples after HTP present notably increased surface area, favoring the adsorption and subsequent hydrolysis by cellulase enzymes. A satisfying enzymatic conversion yield (>85%) at rather low cellulase enzyme dosage (10 FPU/g glucan) was obtained, which would indicate new understandings on the green and efficient bioconversion process on lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

The sugar beet industry is rooted firmly in the trade and agricultural policies of the European powers during the nineteenth century. Disruption of sugar importation into Europe triggered staggering advancements around a home-grown sugar crop based on a newly developed technology out of Lower Silesia. This resulted in today's well-established sugar beet industry that has since spread to all corners of the globe. Over the years, effort has been focused on the improvement of crop output, the reduction of processing costs and energy requirements, as well as the valorization of co-products. Today's industry has started the evolution toward sustainability and a circular economy and is currently gaining momentum. Some high-level scientific research is being transferred to the industry as witness to this process, and it is one of the most exciting times to be part of this realm in Europe. Sugar beet is a near-optimal feedstock for numerous chemical and biochemical products. Not only does it contains an array of valuable and extractable compounds, but it can also serve as a raw material for virtually limitless transformations. This is largely due to accelerating developments in enzymatic, fermentation and synthetic biotechnologies. In a world where sustainability has become a key focus, the sugar beet will continue to play an ever-important role. [ABSTRACT FROM AUTHOR]

The occurrence of emerging contaminants, such as estrogens, in secondary and tertiary treated effluents and in sewage-impacted water bodies is one of the major obstacles to the implementation of water reuse. This review critically evaluates the performance of emerging process of enzymatic degradation of estrogens, and its efficiency. The data collected from peer-review literature show that enzymes have been extensively applied (in both free and immobilized form) in estrogen removal. Amongst others, the use of laccase as a catalyst provides over 90% removal of estrogens. Immobilized enzymes can overcome some limitations of the free biocatalysts, including reusability. Research evidence points to the formation of by-products, such as dimers and trimers. Nevertheless, estrogenic activity assessment indicates a reduction in toxicity after enzyme treatment. The cost and stability of enzymes, as well as their performance in a real wastewater matrix, are the major obstacles to the implementation of enzymatic processes in wastewater treatment. Continued endeavors are required to enhance the successful application of enzymes in the wastewater treatment industry. Processes of enzyme-supported conversion of estrogens are reviewed and discussed. Laccase is the most commonly applied enzyme and achieves over 90% estrogen removal. Immobilization is suggested as an effective tool for enhancement of estrogen removal. Dimers and trimers have been identified as main bioconversion products of estrogens. Existing research gaps are highlighted and future recommendations are provided. [ABSTRACT FROM AUTHOR]

Copyright of Academic Food Journal / Akademik GIDA is the property of Sidas Medya Ajans Tanitim Danismanlik Ltd. Sti and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

BACKGROUND Coffee husk (CH) is the main agricultural solid waste generated during the coffee bean dry processing. Therefore, it is necessary to investigate other alternatives for the more noble use of this by‐product, adding value to it considering the circular economy concept within the coffee production chain. This study aimed to add value to CH by liquid hot water (LHW) pretreatment as an alternative to improve the enzymatic conversion of polysaccharides of this substrate to fermentable sugars through enzymatic hydrolysis. RESULTS: Box–Behnken experimental design was used to evaluate the effect of LHW pretreatment on the enzymatic hydrolysis yield (EHY), investigating the independent variables time (t), temperature (T), and liquid‐to‐solid ratio (LSR). The desirable condition (200 °C, 41 min, LSR of 5 mL g−1) was selected as the most suitable for the enzymatic hydrolysis, reaching an EHY value of 75.3% with an enzyme load of 40 FPU g PCH−1. The alkaline extraction step applied to pretreated coffee husk (PCH) was not effective to improve the EHY, as the cellulose fraction was not preserved. Two reloads of solids were considered an ideal condition, reaching an EHY value of 69.1% after 24 h of hydrolysis with a total enzyme load of 27 FPU g PCH−1. CONCLUSION: The liquid hot water of CHs associated with the reload of solids on enzymatic hydrolysis is an interesting alternative to reduce the cost with enzyme cocktail and hydrolysis time, thus making the process more feasible for technology upscaling. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

The enzymatic conversion of lignins, possibly in combination with electrochemical oxidation, makes aromatics such as syringol, guaiacol, vanillin and catechol available in the qualities required by the fragrance industry. The lignins were obtained by soda digestion from wheat straw and Miscanthus, characterized and then converted with laccases. The overall yield amounted up to 9 wt % with a product spectrum confined to four substances. Catechol was the major product, with a fraction of ≈75 %. It can easily be isolated by extraction with acetone. [ABSTRACT FROM AUTHOR]

Levulinic acid (LA) is an important chemical building block listed among the top 12 value-added chemicals by the United States Department of Energy, and can be obtained through the hydrolysis of lignocellulosic biomass. Using the same approach as in the catalytic production of LA from biomass, catalytic methods to upgrade LA to higher value chemicals have been investigated. Since the discovery of the catabolic genes and enzymes in the LA metabolic pathway, bioconversion of LA into useful chemicals has attracted attention, and can potentially broaden the range of biochemical products derived from cellulosic biomass. With a brief introduction to the LA catabolic pathway in Pseudomonas spp., this review summarizes the current studies on the microbial conversion of LA into bioproducts, including the recent developments to achieve higher yields through genetic engineering of Escherichia coli cells. Three different types of reactions during the enzymatic conversion of LA are also discussed. Key points: • Levulinic acid is an alternative building block to sugars from cellulosic biomass. • Introduction of levulinic acid bioconversion with natural and engineered microbes. • Initial enzymatic conversion of levulinic acid proceeds via three different pathways. • 4-Hydroxyvalerate is one of the target chemicals for levulinic acid bioconversion. [ABSTRACT FROM AUTHOR]

Despite enormous progress in protein engineering, complemented by bioprocess engineering, the revolution awaiting the application of biocatalysis in the fine chemical industry has still not been fully realized. In order to achieve that, further research is required on several topics, including (1) rapid methods for protein engineering using machine learning, (2) mathematical modelling of multi-enzyme cascade processes, (3) process standardization, (4) continuous process technology, (5) methods to identify improvements required to achieve industrial implementation, (6) downstream processing, (7) enzyme stability modelling and prediction, as well as (8) new reactor technology. In this brief mini-review, the status of each of these topics will be briefly discussed. [ABSTRACT FROM AUTHOR]

Background: Conversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route. Results: We summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine (Pinus radiata) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 ±C for 3-72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as evident from the level of retained β-ether interunit linkages, providing an opportunity for conversion into saleable co-products. Conclusions: This process is an efficient route for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. Furthermore, the process uses standard equipment that is largely proven at commercial scale, de-risking process scale-up. [ABSTRACT FROM AUTHOR]

Background: Thermally stable α-L-rhamnosidase with cleaving terminal α-L-rhamnose activity has great potential in industrial application. Therefore, it is necessary to find a proper method to improve the thermal stability of α-L-rhamnosidase. Results: In this study, addition of sorbitol has been found to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus at temperatures ranging from 65 °C to 80 °C. Half-life and activation free energy with addition of 2.0 M sorbitol at 70 °C were increased by 17.2-fold, 8.2 kJ/mol, respectively. The analyses of the results of fluorescence spectroscopy and CD have indicated that sorbitol helped to protect the tertiary and secondary structure of α-L-rhamnosidase. Moreover, the isoquercitrin yield increased from 60.01 to 96.43% with the addition of 1.5 M of sorbitol at 70 °C. Conclusion: Our findings provide an effective approach for enhancing the thermostability of α-L-rhamnosidase from Aspergillus terreus, which makes it a good candidate for industrial processes of isoquercitrin preparation. [ABSTRACT FROM AUTHOR]

An optimal reaction condition for producing cycloamylose (CA) from sweet potato starch was investigated using a combination of isoamylase (from Pseudomonas sp.) and 4-α-glucanotransferase (from Thermus aquaticus, TAαGT). Starch was debranched by isoamylase for 8 h and subsequently reacted with TAαGT for 12 h. The yield and purity of CA products were determined using HPSEC and MALDI-TOFMS, respectively. Consequently, the maximum yield was 48.56%, exhibiting the highest CA production efficiency ever reported from starch. The CA products showed a wide range of the degree of polymerization (DP) with the minimum DP of 5. CA was also produced by simultaneous treatment of isoamylase and TAαGT. The yield was 3.31%, and the final products were contaminated by multiple branched and linear molecules. This result suggests that a former reaction condition (the sequential addition of isoamylase and TAαGT) is preferable for producing CA from sweet potato starch. [ABSTRACT FROM AUTHOR]

The therapeutic efficacy of monoclonal antibodies (mAbs) is dependent on their glycosylation patterns. As the largest group of currently approved biopharmaceuticals, the microheterogeneity in mAb oligosaccharide profiles deriving from mammalian cell production is a challenge to the biopharmaceutical industry. Disengaging the glycosylation process from the cell may offer significant enhancement of product quality and allow better control and reproducibility in line with the Quality-by-Design paradigm. Three potential designs of an Artificial Golgi reactor implementing targeted sequential glycosylation of mAbs are proposed including a (1) microcapillary film reactor, (2) packed bed reactor with nonporous pellets, and (3) packed bed reactor with porous pellets. Detailed mathematical models are developed to predict their performance for a range of design and operational parameters. While all three reactor designs can achieve desired conversion levels, the choice of a particular one depends on the required throughput and the associated cost of enzymes and co-substrates. © 2016 American Institute of Chemical Engineers AIChE J, 62: 2959-2973, 2016 [ABSTRACT FROM AUTHOR]

Cholest-4-en-3-one has positive uses against obesity, liver disease, and keratinization. It can be applied in the synthesis of steroid drugs as well. Most related studies are focused on preparation of cholest-4-en-3-one by using whole cells as catalysts, but production of high-quality cholest-4-en-3-one directly from cholesterol oxidase (COD) using an aqueous/organic two-phase system has been rarely explored. This study set up an enzymatic reaction system to produce cholest-4-en-3-one. We developed and optimized the enzymatic reaction system using COD from COX5-6 (a strain of Rhodococcus) instead of whole-cell biocatalyst. This not only simplifies and accelerates the production but also benefits the subsequent separation and purification process. Through extraction, washing, evaporation, column chromatography, and recrystallization, we got cholest-4-en-3-one with purity of 99.78%, which was identified by nuclear magnetic resonance, mass spectroscopy, and infrared spectroscopy. In addition, this optimized process of cholest-4-en-3-one production and purification can be easily scaled up for industrial production, which can largely decrease the cost and guarantee the purity of the product. [ABSTRACT FROM AUTHOR]

Cephalosporin C (CPC) acylase is an enzyme which hydrolyzes CPC to 7-aminocephalosporanic acid (7-ACA) directly, and therefore has great potential in industrial application. In this study, the CPC acylase from a recombinant Escherichia coli was purified to high purity by immobilized metal affinity chromatography, and the CPC acylase was covalently attached to three kinds of epoxy supports, BB-2, ES-V-1 and LX-1000EP. The immobilized CPC acylase with LX-1000EP as the support shows the highest activity (81 U g) suggesting its potential in industrial 7-ACA production. The activity of immobilized enzyme was found to be optimal at pH between 8.5 and 9.5 and to increase with temperature elevation until 55 °C. Immobilized CPC acylase showed good stability at pH between 8.0 and 9.5 and at temperature up to 40 °C. To avoid product degradation, the production of 7-ACA utilizing immobilized enzyme was carried out at 25 °C, pH 8.5 in a designed reactor. Under optimal reaction conditions, a very high 7-ACA yield of 96.7% was obtained within 60 min. In the results of repeated batch production of 7-ACA, 50% activity of the initial cycle was maintained after being recycled 24 times and the average conversion rate of CPC reached 98%. [ABSTRACT FROM AUTHOR]

Enzymatic removal of blood group A and B antigens from the surface of red blood cells to develop universal blood was a pioneering vision originally proposed more than 25 years ago. A great variety of enzymes, potentially suitable for enzymatic conversion of red blood cells, has been described since, but the process has not been economically viable because of the poor kinetic properties and low pH optimum of enzymes. Recently, the identification of two new families of bacterial glycosidases with enhanced kinetic properties for the removal of A and B antigens at neutral pH marked a milestone in the field of transfusion medicine (Liu et al. 2007). Here we present a detailed structural analysis of Elizabethkingia meningosepticum a-N-acetylgalactosaminidase (NagA) shown to efficiently cleave the A antigen. NagA, a member of glycoside hydrolase (GH) family 109, employs an unusual catalytic mechanism involving NAD+. Comparison of the active-center structure with that of members of GH family 4 reveals a striking degree of structural similarity that allows the postulation of a common reaction mechanism and illustrates a beautiful example of convergent evolution. [ABSTRACT FROM AUTHOR]

The article describes the synthesis of dehydro derivatives of proline-containing cyclic dipeptide by cyclic oxidase and their effects toward cell division. According to the authors, neither of dehydro derivatives inhibited cell division, in contrast to other tetradehydro cyclic dipeptides prepared by a novel enzymatic conversion-guided method, suggesting that an NH proton in a diketopiperazine ring and/or conformation of the compound are important for the activity.

Presents a study on enzymatic synthesis of dehydro cyclo(his-Phe)s, analogs of the potent cell cycle inhibitor, dehydrophenylahistin, and their inhibitory activities toward cell division. Effective conversion of cyclo(His-Phe) to its dehydro derivatives by the enzyme of Streptomyces albulus KO-23; Isolation of two types of dehydro derivatives from the reaction mixture; Findings that indicate an isoprenyl group attached to an imidazole ring of the compound was essential for the inhibitory activity.

The Clostridium perfringens gene encoding the previously characterized α-N-acetylgalactosaminidase (αNAG) was identified by protein microsequencing and database searching. The αNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted α-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic αNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected αNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial αNAG-encoding gene and represents an important step in the development of recombinant αNAG as a tool in the enzymatic conversion of blood group antigens. [Copyright &y& Elsevier]

Copyright of Progress in Modern Biomedicine is the property of Publishing House of Progress in Modern Biomedicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Protoporphyrinogen oxidase, the penultimate enzyme involved in the biosynthetic pathway for heme, catalyzes the removal of six electrons from protoporphyrinogen IX to generate protoporphyrin IX. In Escherichia coli, this enzyme is encoded by the hemG gene. In this study we examined possible alternate pathways for the oxidation of protoporphyrinogen IX to protoporphyrin IX, by isolating and investigating E. coli mutants that can still grow normally when the hemG gene is disrupted. One of these mutants was characterized in detail and had a mutation in the promoter region of the hemF gene, which encodes aerobic coproporphyrinogen oxidase, the enzyme involved in the step immediately before protoporphyrinogen oxidase. Measurement of the promoter activity of the hemF gene showed that the level of transcription was elevated by the mutation. Overexpression of a wild-type hemF gene cloned in a multicopy plasmid also restored the growth of Δ hemG strain. Extracts from cells that overexpress hemF exhibited an increased ability to oxidize protoporphyrinogen IX to protoporphyrin IX. These findings suggest that the E. coli aerobic coproporphyrinogen oxidase has an intrinsic capacity to oxidize not only coproporphyrinogen III but also protoporphyrinogen IX. [ABSTRACT FROM AUTHOR]

Met-Lys-human proinsulin could be converted into insulin in vitro with the treatment of trypsin and carboxypeptides B (CPB). Under less effective conditions, the enzymatic reaction does not proceed perfectly, and twomain bands have been identified by native-polyacrylamide gel electrophoresis (PAGE) analysis. These two main products were thus separated and purified by DEAE-Sephadex A25 chromatography in a Tris-isopropanol system with an NaCl gradient. The isopropanol and NaCl were removed by a second DEAE-Sephadex column. Native-PAGE, mass spectrometric, and amino acid composition analyses indicate that one fraction of these two major products contains human insulin and des B30-insulin and that the other fraction is a mixture of human insulin analogs, which have one more basic amino acid than human in sulin owing to the unsuitable amount of proteases, especially the lack of CPB. Furthermore, both receptor binding assay and radioimmunoassay have been utilized for the activity determination, and both fractions display almost full biological activity with porcine insulin as the standard. Present results provide further evidence for the quality control of recombinant human insulin production. [ABSTRACT FROM AUTHOR]

Applying material balance calculations to the evaluation and optimization of lignocellulosic biomass conversion processes is fundamentally important. The lack of a general framework for material balance calculations and inconsistent compositional analysis data have made it difficult to compare results from different research groups. Material balance templates have been developed to follow accurately the distribution of carbon in lignocellulosic substrates through the pretreatment and simultaneous saccharification and fermentation (SSF) processes, and provide information on overall carbon recovery, recovery of individual sugars, and solubilization of biomass components. Based on material balance considerations, we developed equations that allow us to compute overall ethanol yields for biochemical conversion of biomass correctly. [ABSTRACT FROM AUTHOR]

Partially purified ginsenoside extract (PGE) and compound K enriched extract (CKE) were prepared from ginseng sprouts, and their antioxidant, anti-inflammatory and antithrombotic effects were investigated. Compared to the 6-year-old ginseng roots, ginseng sprouts were found to have a higher content of phenolic compounds, saponin and protopanaxadiol-type ginsenoside by about 56%, 36% and 43%, respectively. PGE was prepared using a macroporous adsorption resin, and compound K(CK) was converted and enriched from the PGE by enzymatic hydrolysis with a conversion rate of 75%. PGE showed higher effects than CKE on radical scavenging activity in antioxidant assays. On the other hand, CKE reduced nitric oxide levels more effectively than PGE in RAW 264.7 cells. CKE also reduced pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β and IL-6 than PGE. Tail bleeding time and volume were investigated after administration of CKE at 70–150 mg/kg/day to mice. CKE administered group showed a significant increase or increased tendency in bleeding time than the control group. Bleeding volume in the CKE group increased than the control group, but not as much as in the aspirin group. In conclusion, ginseng sprouts could be an efficient source of ginsenoside, and CKE converted from the ginsenosides showed antioxidant, anti-inflammatory and antithrombotic effects. However, it was estimated that the CKE might play an essential role in anti-inflammatory effects rather than antioxidant effects. [ABSTRACT FROM AUTHOR]

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The prebiotic potential of longan juice obtained by a commercial Viscozyme L for conversion of constituent sucrose to fructo-oligosaccharide was investigated. The physicochemical properties and carbohydrate composition of the longan juice was evaluated before and after enzymatic treatment. The stimulation effects of the treated longan juice on probiotic bacteria growth were also studied in vitro. The results showed that total soluble solids, yield and clarity of longan juice were all significantly improved after enzyme treatment. The water-soluble polysaccharide content, including pectin, was significantly increased. Compared with the natural longan pulp, the enzyme treated juice showed a significant decrease in sucrose content. Substantial fructo-oligosaccharides including 1-kestose and nystose were synthesized after enzyme treatment. The molecular weight distribution and the monosaccharide composition of the water-soluble polysaccharide were significantly changed by enzyme treatment. The treated longan juice and its ethanol-soluble sugar fraction promoted the growth of Streptococus thermophiles, Lactobacillus acidophilus and Lactobacillus delbrueckii, showing a good potential of the treated longan juice for producing functional foods and nutraceuticals. [ABSTRACT FROM AUTHOR]

In this study, the structural modification of Siam weed (Chromolaena odorata) was performed using NaOH-H2O2- and Ca(OH)2-H2O2-based oxidative pretreatment for the enzymatic conversion of the biomass to a biocommodity, reducing sugar (RS). Pretreatment of raw sample was evaluated at temperatures of 60°C, 70°C, 80°C, 90°C for different time intervals of 3, 6, 9, 12 h in alkaline medium (NaOH or Ca(OH)2). The effects of pretreatment time and temperature were considered in obtaining the optimum conditions. The optimum conditions for NaOH-H2O2 was obtained at 70°C and 3 h with a maximum cellulose content of 44.29%(w/w), lignin content reduced to 21.09% from the initial raw value of 24.2%. Pretreatment with Ca(OH)2-H2O2 resulted in the optimum conditions obtained to be 70°C for 3 h with a cellulose content of 47.18%. Enzymatic hydrolysis on the pretreated biomass at the optimum conditions showed NaOH-H2O2-treated sample yielded 424.35 mg equivalent glucose/g biomass of RS while Ca(OH)2-H2O2-treated sample yielded 335.81 mg equivalent glucose/g biomass of RS. The untreated raw sample yielded 68.75 mg equivalent glucose/g biomass of RS. Consequently, NaOH-H2O2 pretreatment displayed a higher efficiency than Ca(OH)2-H2O2 pretreatment. Stereomicroscopic and scanning electron microscopic imaging of the treated and untreated samples revealed morphological disruptions brought about by the treatments. [ABSTRACT FROM AUTHOR]

Cholest-4-en-3-one has positive uses against obesity, liver disease, and keratinization. It can be applied in the synthesis of steroid drugs as well. Most related studies are focused on preparation of cholest-4-en-3-one by using whole cells as catalysts, but production of high-quality cholest-4-en-3-one directly from cholesterol oxidase (COD) using an aqueous/organic two-phase system has been rarely explored. This study set up an enzymatic reaction system to produce cholest-4-en-3-one. We developed and optimized the enzymatic reaction system using COD from COX5-6 (a strain of Rhodococcus) instead of whole-cell biocatalyst. This not only simplifies and accelerates the production but also benefits the subsequent separation and purification process. Through extraction, washing, evaporation, column chromatography, and recrystallization, we got cholest-4-en-3-one with purity of 99.78%, which was identified by nuclear magnetic resonance, mass spectroscopy, and infrared spectroscopy. In addition, this optimized process of cholest-4-en-3-one production and purification can be easily scaled up for industrial production, which can largely decrease the cost and guarantee the purity of the product. [ABSTRACT FROM AUTHOR]