Enhancing the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase by multi-factors rational design and molecular dynamics simulations.

This study presents a multi-factor rational design strategy combined with molecular dynamics simulation to improve the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase. Candidate mutation sites were identified using Discovery Studio and FoldX software, and the double mutant G124W/G137W was obtained. The mutant was heterogeneously expressed in Escherichia coli strain Rosetta2 (DE3), and its thermostability was verified. Results indicate that the rational design method, combined with molecular dynamics simulation and protein energy calculation, improved the enzyme's thermostability more accurately and effectively. The double mutant G124W/G137W had an optimum temperature of 60°C, about 5.0°C higher than that of the wild-type TYRwt, and its activity was 171.06% higher than the wild-type TYRwt. Its thermostability was enhanced, 42.78% higher than the wild-type at 50°C. These findings suggest that the rational design strategy applied in this study can facilitate the application of industrial enzymes in the pharmaceutical industry.

[Molecular tandem repeat strategy for production ofultrashort peptides].

Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)n. The pET28a-Trxm-(TRSP)n was introduced into E. coli BL21(DE3) to generate a library of Trxm-(TRSP)n sequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)1 achieved 50% of the total protein, while the expression level of Trxm-(TRSP)2 was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)1 were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.

Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli.

A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et  al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

Exploring the Extended Biological Functions of the Human Copper Chaperone of Superoxide Dismutase 1.

The human copper chaperone of SOD1 (designated as CCS) was discovered more than two decades ago. It is an important copper binding protein and a homolog of Saccharomyces cerevisiae LYS7. To date, no studies have systematically or specifically elaborated on the functional development of CCS. This review summarizes the essential information about CCS, such as its localization, 3D structure, and copper binding ability. An emphasis is placed on its interacting protein partners and its biological functions in vivo and in vitro. Three-dimensional structural analysis revealed that CCS is composed of three domains. Its primary molecular function is the delivery of copper to SOD1 and activation of SOD1. It has also been reported to bind to XIAP, Mia40, and X11α, and other proteins. Through these protein partners, CCS is implicated in several vital biological processes in vivo, such as copper homeostasis, apoptosis, angiogenesis and oxidative stress. This review is anticipated to assist scientists in systematically understanding the latest research developments of CCS for facilitating the development of new therapeutics targeting CCS in the future.

Enzymatic synthesis of cefazolin using immobilized recombinant cephalosporin-acid synthetase as the biocatalyst.

A method for the synthesis of ß-lactam antibiotic cefazolin (CEZ) by enzymatic acylation of 7-amino-3-(5-methyl-l,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (TDA) using immobilized cephalosporin-acid synthetase (IECASA) from recombinant E. coli strain VKPM B-12316 has been developed. A stepwise pH gradient designed on the basis of investigations on the solubility of components was applied for synthesis. This helped in avoiding the precipitation of TDA in the reaction when its initial concentration was high (150-200 mM). Thus, under optimal conditions a high yield of CEZ (relative to TDA) of 92-95% was obtained. Where the final reaction mixture contained 65-85 mg/mL of CEZ, 4-5 mg/mL of unreacted TDA, and 40-60 mg/mL of the by-product, 1(H)-tetrazolylacetic acid (TzAA). Testing of optimized CEZ synthesis using IECASA in a batch reactor has proved sufficiently high operational stability of the biocatalyst, with its residual activity after the 25th cycle accounting for about 83 ± 2% of its starting value. The half-inactivation period of IECASA was estimated as 85 cycles of CEZ synthesis.

Inadequate weight gain in obese women and the risk of small for gestational age (SGA): a systematic review and meta-analysis.

OBJECTIVES: To examine the association between small for gestational age (SGA) and inadequate gestational weight gain (GWG) in obese women (compared with Institute of Medicine [IOM] guidelines) stratified by obesity classes. METHODS: We conducted a meta-analysis of original researches with sufficient information about inadequate GWG in obese women stratified by obesity classes. SGA as the chief outcome was extracted and assessed in our analysis. MEDLINE and EMBASE were searched through Ovid from 28 May 2009 to 1 December 2015. Quality was assessed using a modified Newcastle-Ottawa scale. RESULTS: 480 citations were screened and 13 studies (437 512 obese women) were included. Obese women who gained weight below the guidelines had higher risks of SGA than those who gained weight within the guidelines (OR 1.28; 95% CI 1.14-1.43). The same conclusions were also confirmed in Class I, Class II and Class III of obese women: Class I (OR 1.37; 95% CI 1.22-1.54); Class II (OR 1.38; 95% CI 1.24-1.54); Class III (OR 1.25; 95% CI 1.14-1.36). CONCLUSIONS: From our analysis, the guidelines of IOM can be applied to all the classes of obesity. More accurate boundaries for each obesity class should be established to evaluate the maternal and fetal risks. Diverse populations are thus necessary for more studies in the future.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.

[Synthesis of cefatrizine by recombinant alpha-amino acid ester hydrolase].

To explore the enzymatic route of cefatrizine synthesis, alpha-amino acid ester hydrolase (AEH) gene was cloned from the whole genome of Xanthomonas rubrillineans, and expressed heterologously in Escherichia coli BL21 (DE3). The effects of temperature, pH and substrates' molar ratio upon the transformation yield of cefatrizine by purified recombinant AEH were investigated. The monomer of AEH was determined as 70 kDa by SDS-PAGE. The optimal pH and temperature reaction were (6.0 +/- 0.1) and 36 degrees C for cefatrizine synthesis. The transformation yield was 64.3% under 36 degrees C, pH (6.0 +/- 0.1), when the concentrations of two substrates were about 30 mmol/L (7-ATTC) and 120 mmol/L (HPGM x HCl), respectively, and the enzyme consumption was 22 U/mL. The results pave the way for optimization of the industrial enzymatic synthesis of cefatrizine.

[Clinical study on simvastatin treatment of pulmonary hypertension in patients with coal worker's pneumoconiosis].

OBJECTIVE: To observe simvastatin treatment of pulmonary hypertension in patients with coal workers pneumoconiosis (CWP). METHODS: 96 CWP patients with pulmonary hypertension were randomly divided into treatment and control groups. The control group was treated with 2.5 mg warfarin, once a day for four months; the treatment group was treated with 20 mg simvastatin, taken in evening, for 4 months. 6 min walking distance (6MWD) test and inspection pulmonary artery pressure were measured by echocardiography before and after treatment. RESULTS: In the treatment group, the 6MWD were (258 ± 26) m after treatment and (225 ± 19) m before treatment, respectively. Compared with control group, pulmonary artery pressure was (41 ± 9) mm Hg in the treatment group before treatment, (36 ± 3) mm Hg in the treatment group after treatment, and (39 ± 5) mm Hg in control group, respectively, the difference was statistically significant (P < 0.05). CONCLUSIONS: Simvastatin can improve pulmonary hypertension in coal workers pneumoconiosis, and shows a definite curative effect.