High-Level Production of Recombinant Lipase by Fed-Batch Fermentation in Escherichia coli and Its Application in Biodiesel Synthesis from Waste Cooking Oils.

The enzymatic production of biodiesel from waste cooking oils (WCOs) offers a green and sustainable solution for the liquid fuel manufacture as well as waste resource recovery. In present study, liquid lipase was used to simplify the catalysis process, thereby reducing biodiesel production costs. An engineered Escherichia coli expressing Geobacillus thermocatenulatus lipase 2 (GTL2) was screened at an enzyme activity of 6.96 U/mg, after evaluating the propagating stability of the recombinant plasmids exceeding 86.11%. Through the beneficial feeding strategy and effective pH control, high-level production of GTL2 by fed-batch fermentation was achieved with an enzyme activity of 434.32 U/mg, which was almost 62 times that of shake flask fermentation. In addition, liquid GTL2 was used to prepare fatty acid methyl esters (FAMEs) using WCOs. The effects of the reaction time, catalyst loading, temperature, and methanol-to-oil molar ratio on FAMEs production using WCOs were explored, and a maximum FAMEs yield of 96.62% was achieved under optimized conditions. These results indicate that liquid GTL2 is a promising biocatalyst for efficient utilization of WCOs in the synthesis of biodiesel and provide a novel enzymatic process for biodiesel reducing the cost of production.

N-glycosylation as an effective strategy to enhance characteristics of Rhizomucor miehei lipase for biodiesel production.

The construction of methanol-resistant lipases with high catalytic activity is world-shattering for biodiesel production. A semi-rational method has been constructed to enhance the properties of Rhizomucor miehei lipase with propeptide (ProRML) by introducing N-glycosylation sites in the Loop structure. The enzyme activities of the mutants N288 (1448.89 ± 68.64 U/mg) and N142 (1073.68 ± 33.87 U/mg) increased to 56.09 and 41.56 times relative to that of wild type ProRML (WT, 25.83 ± 0.73 U/mg), respectively. After incubation in 50 % methanol for 2.5 h, the residual activities of N314 and N174-1 were 95 % and 85%, which were higher than the WT (27 %). Additionally, the biodiesel yield of all mutants was increased after a one-time addition of methanol for 24 h. Among them, N288 increased the quantity of biodiesel from colza oil from 9.49 % to 88 %, and N314 increased the amount of biodiesel from waste soybean oil from 8.44% to 70%. This study provides an effective method to enhance the properties of lipase and improve its application potential in biodiesel production.

Isolation, identification, and whole-genome sequencing of high-yield protease bacteria from Daqu of ZhangGong Laojiu.

A total of 296 strains of protease-producing bacteria were isolated and purified from medium-temperature Daqu produced by ZhangGong LaoJiu Wine Co. Ltd. After calculating the ratio of transparent ring diameter to colony diameter and measuring the protease activities, a strain of high-yield protease bacteria, called DW-7, was screened out with a protease activity of 99.54 U/mL. Through morphological observation, 16S rDNA sequence analysis, and physiological and biochemical tests, the isolated bacteria DW-7 was determined to be Bacillus velezensis. In addition, whole-genome sequencing (WGS), using PacBio and the Illumina platform, was performed. Gene annotation was then conducted using the Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Non-Redundant Protein Sequence Database (NR), and Gene Ontology (GO) databases. The results showed that the genome of DW-7 was 3,942,829 bp long with a GC content of 46.45%. A total of 3,662 protein-encoding genes were predicted, with a total length of 3,402,822 bp. Additionally, 2,283; 2,796; and 2,127 genes were annotated in the COG, KEGG, and GO databases, respectively. A total of 196 high-yield protease genes were mainly enriched in the metabolism of alanine, aspartic acid, glutamate, glycine, serine, and threonine, as well as ABC transporter and transporter pathways.

Improvement of methanol tolerance and catalytic activity of Rhizomucor miehei lipase for one-step synthesis of biodiesel by semi-rational design.

Exploiting highly active and methanol-resistant lipase is of great significance for biodiesel production. A semi-rational directed evolution method combined with N-glycosylation is reported, and all mutants exhibiting higher catalytic activity and methanol tolerance than the wild type (WT). Mutant N267 retained 64% activity after incubation in 50% methanol for 8 h, which was 48% greater than that of WT. The catalytic activity of mutants N267 and N167 was 30- and 71- fold higher than that of WT. Molecular dynamics simulations of N267 showed that the formation of new strong hydrogen bonds between glycan and the protein stabilized the structure of lipase and improved its methanol tolerance. N267 achieved biodiesel yields of 99.33% (colza oil) and 81.70% (waste soybean oil) for 24 h, which was much higher than WT (51.6% for rapeseed oil and 44.73% for wasted soybean oil). The engineered ProRML mutant has high potential for commercial biodiesel production.

Improved methanol tolerance of Rhizomucor miehei lipase based on N­glycosylation within the α-helix region and its application in biodiesel production.

BACKGROUND: Liquid lipases are widely used to convert oil into biodiesel. Methanol-resistant lipases with high catalytic activity are the first choice for practical production. Rhizomucor miehei lipase (RML) is a single-chain α/ß-type protein that is widely used in biodiesel preparation. Improving the catalytic activity and methanol tolerance of RML is necessary to realise the industrial production of biodiesel. RESULTS: In this study, a semi-rational design method was used to optimise the catalytic activity and methanol tolerance of ProRML. After N-glycosylation modification of the α-helix of the mature peptide in ProRML, the resulting mutants N218, N93, N115, N260, and N183 increased enzyme activity by 66.81, 13.54, 10.33, 3.69, and 2.39 times than that of WT, respectively. The residual activities of N218 and N260 were 88.78% and 86.08% after incubation in 50% methanol for 2.5 h, respectively. In addition, the biodiesel yield of all mutants was improved when methanol was added once and reacted for 24 h with colza oil as the raw material. N260 and N218 increased the biodiesel yield from 9.49% to 88.75% and 90.46%, respectively. CONCLUSIONS: These results indicate that optimising N-glycosylation modification in the α-helix structure is an effective strategy for improving the performance of ProRML. This study provides an effective approach to improve the design of the enzyme and the properties of lipase mutants, thereby rendering them suitable for industrial biomass conversion.

Enhanced activity of Rhizomucor miehei lipase by directed saturation mutation of the propeptide.

The propeptide is a short sequence that facilitates protein folding. In this study, four highly active Rhizomucor miehei lipase (RML) mutants were obtained through saturation mutagenesis at three propeptide positions: Ser8, Pro35, and Pro47. The enzyme activities of mutants P35 N, P47 G, P47 N, and S8E/P35S/P47A observed at 40 °C, and pH 8.0 were 10.19, 7.53, 6.15, and 8.24 times of that wild-type RML, respectively. The S8E/P35S/P47A mutant showed good thermostability. After incubation at 40 °C for 1 h, 98.98 % of its initial activity remained, whereas wild-type RML retained only 78.76 %. This result indicated that the enhancement of hydrophilicity of 35- and 47- amino-acid residues could promote the interaction between the propeptide and the mature peptide and the enzyme activity and expression level. Highly conserved sites had a more significant impact on enzyme performance than did other sites, similar to the Pro35 and Pro47 mutants showed in this study. This study provides a new idea for protein modification: enzyme performance can be improved through propeptide regulation.

Crude glycerol impurities improve Rhizomucor miehei lipase production by Pichia pastoris.

Crude glycerol, a by-product of biodiesel production, was employed as the carbon source to produce lipase using Pichia pastoris. Under identical fermentation conditions, cell growth and lipase activity were improved using crude glycerol instead of pure glycerol. The impacts of crude glycerol impurities (methyl ester, grease, glycerol, methanol, and metal ions Na+, Ca2+, and Fe3+) on lipase production were investigated. Impurities accelerated P. pastoris entering the stationary phase. Na+, Ca2+, and grease in waste crude glycerol were the main factors influencing higher lipase activity. Through response surface optimization of Ca2+, Na+, and grease concentrations, lipase activity reached 1437 U/mL (15,977 U/mg), which was 2.5 times that of the control. This study highlights the economical and highly efficient valorization of crude glycerol, demonstrating its possible utilization as a carbon source to produce lipase by P. pastoris without pretreatment.

[Propeptide-mediated protein folding: mechanism and its impact on lipase].

The formation of most proteins consists of two steps: the synthesis of precursor proteins and the synthesis of functional proteins. In these processes, propeptides play important roles in assisting protein folding or inhibiting its activity. As an important polypeptide chain coded by a gene sequence in lipase gene, propeptide usually functions as an intramolecular chaperone, assisting enzyme molecule folding. Meanwhile, some specific sites on propeptide such as glycosylated sites, have important effect on the activity, stability in extreme environment, methanol resistance and the substrate specificity of the lipase. Studying the mechanism of propeptide-mediated protein folding, as well as the influence of propeptide on lipases, will allow to regulate lipase by alternating the propeptide folding behavior and in turn pave new ways for protein engineering research.

Novel Sanger's Reagent-like Styrene Polymer for the Immobilization of Burkholderia cepacia Lipase.

Sanger's reaction, which was originally developed for amino acid detection, was utilized for enzyme immobilization. The newly synthesized polymer support, which was called polymer NO2-4-fluorostyrene-divinylbenzene (pNFD), was embedded with a Sanger's reagent-like functional group for immobilizing enzymes covalently under mild reaction conditions. Using Burkholderia cepacia lipase (BCL) as the target enzyme, the immobilization efficiency and activity of pNFD-BCL reached as high as 1.2 mg·g-1 and 33.21 U·g-1 (a specific activity of 27 675 U·g-1), respectively, realizing 90% activity recovery. It also improved the optimal reaction temperature of BCL from 40 to 65 °C, under which its full activity could be retained for 4 h. The new carrier also widened the pH-adaptive range of BCL as 6.5-10.0, allowing the lipase to operate normally in weak acid environment. Reusability of pNFD-BCL was significantly improved as almost no activity and/or enantioselectivity loss was observed in 200 h of triglyceride hydrolysis reaction and 17 batches of ( R, S)-1-phenylethanol resolution reaction.

BIBLIOMETRIC ANALYSIS OF ACUPUNCTURE RESEARCH FRONTS AND THEIR WORLDWIDE DISTRIBUTION OVER THREE DECADES.

BACKGROUND: Considerable research has been conducted on acupuncture worldwide. This study chronologically examined the changing features and research fronts of acupuncture and elucidated the differences among the six most productive countries. METHODS: Bibliographic coupling is a powerful tool for identifying the research fronts of a field. Acupuncture-related publications worldwide and from the six most productive countries during 1983-2012 were retrieved from the Science Citation Index Expanded and Social Science Citation Index. To form the research fronts, the 100 most highly cited papers (HCPs) were clustered in terms of references shared. RESULTS: The United States had the highest proportion of HCPs. The effectiveness of acupuncture in areas such as relieving neck and back pain, migraines and headaches, and knee osteoarthritis symptoms was a predominant topic. Initially, the endogenous opioid peptide system was the primary research focus in the acupuncture mechanism research; however, during 1993-2012, researchers focused more on the functional magnetic resonance imaging of brain activity. In addition, acupuncture use and prevalence, the attitudes of health practitioners, and the effects of expectancy and belief were also major topics. Researches from Western countries, including the United States, England, and Germany, showed more interest in clinical trials and economic- and ethics-related studies, whereas those from East Asian countries including China, Japan, and South Korea focused more on mechanism research. CONCLUSION: Western countries dominated the research fronts of acupuncture. The patterns of the research fronts varied worldwide, indicating continuity and innovation in research in each country.

One-step hydroprocessing of fatty acids into renewable aromatic hydrocarbons over Ni/HZSM-5: insights into the major reaction pathways.

For high caloricity and stability in bio-aviation fuels, a certain content of aromatic hydrocarbons (AHCs, 8-25 wt%) is crucial. Fatty acids, obtained from waste or inedible oils, are a renewable and economic feedstock for AHC production. Considerable amounts of AHCs, up to 64.61 wt%, were produced through the one-step hydroprocessing of fatty acids over Ni/HZSM-5 catalysts. Hydrogenation, hydrocracking, and aromatization constituted the principal AHC formation processes. At a lower temperature, fatty acids were first hydrosaturated and then hydrodeoxygenated at metal sites to form long-chain hydrocarbons. Alternatively, the unsaturated fatty acids could be directly deoxygenated at acid sites without first being saturated. The long-chain hydrocarbons were cracked into gases such as ethane, propane, and C6-C8 olefins over the catalysts' Brønsted acid sites; these underwent Diels-Alder reactions on the catalysts' Lewis acid sites to form AHCs. C6-C8 olefins were determined as critical intermediates for AHC formation. As the Ni content in the catalyst increased, the Brønsted-acid site density was reduced due to coverage by the metal nanoparticles. Good performance was achieved with a loading of 10 wt% Ni, where the Ni nanoparticles exhibited a polyhedral morphology which exposed more active sites for aromatization.

[A bibliometric overview of traditional Chinese medicine research in Medline].

OBJECTIVE: To analyze the publication activity in traditional Chinese medicine (TCM) and learn more about the development and perspective of TCM as well as its subfields. METHODS: TCM-related publications (from 1964 to 2008) indexed in Medline were analyzed, including publication number and proportion of TCM papers, TCM clinical research papers, papers in TCM subfields such as Chinese medicine, Chinese herbs and acupuncture. We also analyzed the journals in which they got published. RESULTS: A total of 34 528 TCM-related papers were indexed in Medline from 1964 to 2008. The number and proportion of publication in Medline were increased steadily. TCM articles have changed towards clinical-oriented research, especially in complementary therapies and acupuncture subfields. Before 1973 the popular content of TCM articles was acupuncture, but at present it changed to Chinese herbs. TCM papers were found in core clinical journals, specialized complementary journals and non-core journals, the last one was the majority. CONCLUSION: The publication activity of TCM literature increased rapidly since 1970s. TCM attracts more and more attentions and researchers. The safety and efficacy of TCM have been tried to identify by conducting modern scientific researches.

[Current status of research on complementary and alternative medicine in the United States: in comparison with that on traditional Chinese medicine in China].

Researches on complementary and alternative medicine (CAM), including traditional Chinese medicine (TCM), are attracting more and more attention in the United States, and their scale and investment are progressively increasing. The differences between China and the United States in application of TCM therapies, background of Eastern and Western knowledge, thinking model, as well as the recognition of TCM theories lead to the differences in content and design of TCM researches.

[Analysis of international application of complementary and alternative medicine].

Traditional Chinese medicine (TCM) is an important part of complementary and alternative medicine (CAM) in the world medical market. The basis of globalization of TCM was to make sure the current situation of CAM and the role that TCM plays. It will improve the application and reputation of TCM by analyzing the global usage rate, sort of diseases, social-demographic characteristics of users and medical economics of CAM.