An Approach to the Production of Soluble Protein from a Fungal Gene Encoding an Aggregation-Prone Xylanase in Escherichia coli

The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ32). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by σ32. One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of σ32. The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes

Efficacy mechanisms research progress of the active components in the characteristic woody edible oils

Woody edible oils are a type of vegetable oil. Woody edible oils like olive oil have greater quantities of unsaturated fatty acids (UFAs), particularly essential FAs, as well as vitamin E, phytosterols, and other nutrients that are becoming more vital in human health. As a result, finding high-quality woody oil resource plants is critical to ensuring enough edible oil supply. As six novel woody crops, Paeonia suffruticosa, Plukenetia volubilis, Acer truncatum, Olea europaea, Camellia sinensis, and Camellia oleifera are characterized by high oil production, widespread cultivation, adaptability, and various active ingredients. The six woody crop oils contain UFAs (e.g., α-linolenic acid, oleic acid, and linoleic acid), vitamin E, polyphenols, phytosterols, and so forth. The presence of these active ingredients confers anti-inflammatory, antioxidant, cholesterol and lipid metabolism regulating, blood lipid lowering, immune boosting, memory improving, intestinal flora regulating, and other properties to the oils, which are beneficial to body health. This article examined in depth the seed resources, FA composition, active component kinds, active ingredient efficacy mechanism, and physiological impacts of these six novel woody crop oils. These developments lay a solid platform for further study and development of these woody oil crops.This work was supported by the Key Research and Development Program of Zhejiang Province (No. 2021C02002), Zhejiang Provincial Natural Sciences Foundation of China under Grant (No. LZ22C200006), Top young talents of the ten thousand talents program of Zhejiang Province (ZJWR0308016), Key R&D projects in Zhejiang Province (2023C04010), and Zhejiang Basic Public Welfare Research Project (LGN21C200006). Agusti Romero acknowledges financial support from the CERCA Program from the Generalitat of Catalonia. We would like to thank all contributors of the current study for their concepts, ideas, contribution, and provision.info:eu-repo/semantics/publishedVersio

Identification of Genes Directly Involved in Shell Formation and Their Functions in Pearl Oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the ‘aragonitic line’. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P.fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the ‘aragonitic line’, and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Study QoS Optimization and Energy Saving Techniques in Cloud, Fog, Edge, and IoT

With an increase of service users’ demands on high quality of services (QoS), more and more efficient service computing models are proposed. The development of cloud computing, fog computing, and edge computing brings a number of challenges, e.g., QoS optimization and energy saving. We do a comprehensive survey on QoS optimization and energy saving in cloud computing, fog computing, edge computing, and IoT environments. We summarize the main challenges and analyze corresponding solutions proposed by existing works. This survey aims to help readers have a deeper understanding on the concepts of different computing models and study the techniques of QoS optimization and energy saving in these models

Orlistat response to missense mutations in lipoprotein lipase

The human lipoprotein lipase (LPL) is a therapeutic target for obesity, and inhibition of LPL with the approved small molecule agent orlistat has been widely used in clinic to treat obesity-related health problems such as diabetes and cardiovascular diseases. However, a variety of missense mutations in LPL protein have been observed, which may cause resistance or sensitization for orlistat, largely limiting the clinical applications of orlistat in obesity therapy. Here, we integrated molecular dynamics simulations and enzyme inhibition to investigate orlistat response to 16 disorder-associated missense mutations in LPL catalytic domain. It was found that most mutations have a modest effect on orlistat binding, and only few can exert strong impact to the binding. Three unfavorable (Trp86Arg, Ile194Thr, and Glu242Lys) and two favorable (His136Arg and Gly188Glu) mutations were identified, which can alter the binding affinity and inhibitory activity of orlistat considerably. Structural and energetic analysis revealed that these potent mutations induce orlistat resistance and sensitization by directly influencing the intermolecular interaction between LPL and orlistat or by indirectly addressing allosteric effect on LPL structure. (C) 2016 International Union of Biochemistry and Molecular Biology, Inc.</p

Impact of orientation and flexibility of peptide linkers on T-maritima lipase Tm1350 displayed on Bacillus subtilis spores surface using CotB as fusion partner

Fusion protein construction often requires peptide linkers for prolonged conformation, extended stability and enzyme activity. In this study a series of fusion between Thermotoga maritima lipase Tm1350 and Bacillus subtillis coat protein CotB, comprising of several peptide linkers, with different length, flexibility and orientations were constructed. Effects of temperature, pH and chemicals were examined, on the activity of displayed enzyme. The fusion protein with longer flexible linkers L9 [(GGGGS)(4)] and L7 (GGGGS-GGGGS-EAAAK-EAAAK-GGGGS-GGGGS) possess 1.29 and 1.16-fold higher activity than the original, under optimum temperature and pH respectively. Moreover, spore surface displaying Tm1350 with L3 (EAAAK-GGGGS) and L9 ((GGGGS) 4) showed extended thermostably, maintaining 1.40 and 1.35-fold higher activity than the original respectively, at 80 degrees C after 5 h of incubation. The enzyme activity of linkers with different orientation, including L5, L6 and L7 was determined, where L7 maintained 1.05 and 1.27-fold higher activity than L5 and L6. Effect of 0.1% proteinase K, bromelain, 20% ethanol and 30% methanol was investigated. Linkers with appropriate Glycine residues (flexible) showed higher activity than Alanine residues (rigid). The activity of the displayed enzyme can be improved by maintaining orientation and flexibility of peptide linkers, to evaluate high activity and stability in industrial processes.</p

Reliability Analysis on One-Step Overall Transportation of Composite Bucket Foundation for Offshore Wind Turbine

Composite bucket foundations, which have been successfully transported, installed, and operated at the Qidong, Xiangshui, and Dafeng offshore wind farms in China, are economically advantageous due to the relatively simple transportation and installation process. The innovative one-step transportation and installation technology of foundation-tower-nacelle is the key phase in saving costs. In this paper, a &ldquo;foundation lift ship&rdquo; overall transport mode is proposed and introduced for the first time. Prototype data measurement, preliminary numerical simulation, and theoretical calculations were conducted to investigate whether the foundation-ship integrity, tower hoop stability, and various indexes of the nacelle met the requirements under the influences of various environmental factors. The multi-system coupling motion mechanism and analysis method of this new structure and transportation mode were expounded. Through the prototype observation data of the one-step overall transportation, the ship-foundation system reliability of the structure in the case of large wind and wave was confirmed. Furthermore, it was found that in the one-step overall transportation, the importance of factors to nacelle acceleration decreased in the order of wave height, current speed, and wind speed by the time and frequency domain analysis and data statistics

Clostridium thermocellum Nitrilase Expression and Surface Display on Bacillus subtilis Spores

Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from Clostridium thermocellum was expressed, identified and displayed on the surface of Bacillus subtilis spores by using the spore coat protein G of B. subtilis as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40 degrees C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the sporeimmobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion. (C) 2015 S. Karger AG, Basel</p