In vivo self-aggregation and efficient preparation of recombinant lichenase based on ferritin / 生物工程学报

Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 ℃ was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.

Advances of enzymes in the applications of disease treatment and drug preparation / 生物工程学报

The development of biotechnology and the in-depth research on disease mechanisms have led to increased application of enzymes in the treatment of diseases. In addition, enzymes have shown great potential in drug manufacturing, particularly in production of non-natural organic compounds, due to the advantages of mild reaction conditions, high catalytic efficiency, high specificity, high selectivity and few side reactions. Moreover, the application of genetic engineering, chemical modification of enzymes and immobilization technologies have further improved the function of enzymes. This review summarized the advances of using enzymes as drugs for disease treatment or as catalysts for drug manufacturing, followed by discussing challenges, potential solutions and future perspectives on the application of enzymes in the medical and pharmaceutical field.

Mining and engineering of microbial carbonic anhydrases for biomimetic carbon dioxide sequestration / 生物工程学报

The increasing atmospheric carbon dioxide levels have been correlated with global warming. Carbonic anhydrases (CA) are the fastest among the known enzymes to improve carbon capture. The capture of carbon dioxide needs high temperature and alkaline condition, which is necessary for CaCO₃ precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential, and polyextremophilic microbes are one of the important sources of these enzymes. The current review focuses on both those isolated by thermophilic organisms from the extreme environments and those obtained by protein engineering techniques, and the industrial application of the immobilized CAs is also briefly addressed. To reduce the greenhouse effect and delay global warming, we think further research efforts should be devoted to broadening the scope of searching for carbonic anhydrase, modifying the technology of protein engineering and developing highly efficient immobilization strategies.

Oligomerization triggered by foldon to enhance the catalytic efficiency of feruloyl esterase / 生物工程学报

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.

Effect of microRNA-152 on cholesterol accumulation in RAW264.7 and identification of its functional tar-get genes / 实用医学杂志

Objective To investigate the effect of miR-152 on ox-LDL induced cholesterol accumulation in RAW264.7 macrophages and to verify its target genes. Methods RAW264.7 macrophages were divided into two groups:miR-152 group and negative control group. Total cholesterol(TC),cholesterol ester(CE)concentra- tion and the ratio of CE/TC were observed in the two group by ox-LDL for 48 h. Furthermore,bioinformatics meth-od,dual luciferase reporter assay,real-time quantitative PCR and Western blot were applied to detect the target gene of miR-152. Results As compared with the control group,the contents of TC,CE and the ratio of CE/TC were increased in the miR-152 group. The mRNA and protein expressions of ABCA1 were significantly lower in miR-152 group. Conclusions MiR-152 could inhibit macrophage foam cell formation through decreasing the expres-sion of ABCA1.

Effect of microRNA-152 on cholesterol accumulation in RAW264.7 and identification of its functional tar-get genes / 实用医学杂志

Objective To investigate the effect of miR-152 on ox-LDL induced cholesterol accumulation in RAW264.7 macrophages and to verify its target genes. Methods RAW264.7 macrophages were divided into two groups:miR-152 group and negative control group. Total cholesterol(TC),cholesterol ester(CE)concentra- tion and the ratio of CE/TC were observed in the two group by ox-LDL for 48 h. Furthermore,bioinformatics meth-od,dual luciferase reporter assay,real-time quantitative PCR and Western blot were applied to detect the target gene of miR-152. Results As compared with the control group,the contents of TC,CE and the ratio of CE/TC were increased in the miR-152 group. The mRNA and protein expressions of ABCA1 were significantly lower in miR-152 group. Conclusions MiR-152 could inhibit macrophage foam cell formation through decreasing the expres-sion of ABCA1.

Advances in protein cyclization / 生物工程学报

Proteins, which exist mainly in linear form in vivo, are easily affected by the change of ambient temperature and pH. The application of proteins (enzymes) in the fields of industrial catalyzing, food manufacturing and medicine are restricted due to their properties. The cyclic structure of natural cyclic peptides confers high thermal stability on itself; such mechanism can be referred to in further enhancement of the thermal stability and transformation of the structure of enzymes. This article reviewed the latest progress in the domestic and international studies on protein cyclization and summarized the traditional methods (such as protein trans-splicing, expressed protein ligation and sortase-catalyzed transpeptidation) in protein cyclization. A novel method based on SpyTag/SpyCather-mediated enzyme cyclization was discussed in more detail.

Progress in co-immobilization of multiple enzymes / 生物工程学报

Enzyme immobilization is the core technology of biocatalysis. Over the past few decades, enzyme immobilization research mainly focused on single enzyme immobilization. In recent years, multi-enzyme immobilization attracts more and more attention as it could increase the local concentration of reaction and improve the reaction yield. In this review, a summary of the recent progress, together with our research, is presented. Special emphasis is placed on four methods in multi-enzymes co-immobilization, namely, the nonspecific covalent co-immobilization, the nonspecific non-covalent co-immobilization, the non-covalent encapsulation co-immobilized and the site specificity co-immobilized. Finally, some industrial uses of immobilized multi-enzymes were addressed and the application prospect of multi-enzyme immobilization was highlighted.

Factors and mechanism influencing elastin-like polypeptides self-assembled into micron-sized particles / 生物工程学报

Many factors influence the elastin-like polypeptides (ELPs) self-assembled into micron-sized particles. However, few efforts were made to investigate these factors. Using the ELPs [KV8F]n as the target, we studied systematically the factors with the dynamic light scattering. Our results show that the particle size increased and the uniform of particles decreased with the increase of the molecular weight. The analysis of size variation in self-assembled ELPs in response to changes in salt concentration indicated that the size increased with increasing the salt concentration, and the opposite response was observed when the concentration was above 0.4 mol/L. Under these conditions, the particles are micron-sized and larger than 1.1 μm. However, when the fusions containing the same ELPs and xylanase or 1,3-propanediol oxidoreductase, the size of the self-assembled ELPs particles decreased dramatically, which was only about 1/10 of that of the free ELPs. We proposed that the solvent accessible charged area of the enzymes could interact with the ELPs, the sterical hindrance of the enzymes prevent the aggregation of the ELPs. This might be the most important parameter in altering the particle size sharply.

Application of pancreaticojejunostomy procedures selection strategy in pancreaticoduodenectomy / 中华消化外科杂志

Objective To evaluate the pancreaticojejunostomy procedures selection strategy in pancreaticoduodenectomy.Methods The clinical data of 455 patients who received pancreaticoduodenectomy at the Xijing Hospital from June 2007 to June 2012 were retrospectively analyzed.For patients with pancreatic duct diameter≥4 mm,duct-to-mucosa pancreaticojejunostomy(DMPJ)was applied(DMPJ group,210 cases).For patients with pancreatic duct diameter ＜ 4 mm,modified Child pancreaticojejunostomy was applied to 140 patients(modified Child group)whose jejunal end was smaller than the pancreatic stump,and binding pancreaticojejunostomy was applied to 105 patients(binding group)whose jejunal end was bigger than or equal to the pancreatic stump.The clinical efficacy and incidence of postoperative complications were compared among the 3 groups.The count data and measurement data were analyzed by chi-square test and t test,respectively.Results The pancreatic duct diameter of the DMPJ group was(4.4 ± 0.7)mm,which was significantly bigger than(2.8 ± 0.6)mm of the modified Child group and(2.3 ± 0.7)mm of the binding group(t =2.25,2.48,P ＜ 0.05).The diameter of the pancreatic stump of the modified Child group was(36 ± 5)mm,which was significantly bigger than(21 ± 6)mm of the binding group(t =21.65,P ＜ 0.05).The overall incidence of pancreatic leakage was 8.4％(38/455).There were no significant differences in the incidences of pancreatic leakage,peritoneal bleeding,abdominal infection,digestive dysfunction rate and the mean duration of hospital stay among the 3 groups(x2 =0.53,0.88,1.63,5.34,F =2.53,P ＞ 0.05).Conclusion Pancreaticojejunostomy procedure selection strategy based on the diameters of pancreatic duct and pancreatic stump could obtain good clinical efficacy and is appropriate.

De novo design, non-chromatographic purification and salt-effect of elastin-like polypeptides / 生物工程学报

Elastin-like polypeptides (ELPs) are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin. It was a promising tag for recombinant protein purification. Here, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b(+). Then, we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl beta-D-Thiogalactoside (IPTG), ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the [KV8F-20] decreased to 19 degrees C by 0.4 mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.

Study on the correlation between the anti-earthquake military rescuers' psychosomatic health and social support and mental intervention / 中华行为医学与脑科学杂志

Objective To investigate the relationship between the anti-earthquake military rescuers' psychosomatic health and social support, and observe the effect of intervention to the army men who suffered troubles.Methods By cluster random sampling, 152 military rescuers and 139 army men not taking part in the rescues were investigated by Facility Psychosomatic Health Scale and Social Support Rating Scale. Mental intervention or medication was performed to those produced psychosomatic health. Results The scores of somatic symptoms,mental status,psychosomatic health and social support of military rescuers and the control group were (4.09±4.70) ,(2.18 ±3.16) ,(6.27 ±7.08) ,(6.14 ±4.20) and (2.11 ±4.16) ,(1.37 ±3.09),(3.47 ±6.55),(4.57 ± 4.17 )respectively, the difference was significant(P＜0.05). The somatic symptoms had significantly positive correlation with mental status in military rescuers (r=0.17～0.52, P＜ 0.05 or 0.01 ). Failure of social support had positive correlation with mental disorders (r=0.18～0.40, P＜0.05 or 0.01). The degree of social support was the main factor which had affected on the psychosomatic health of military rescuers. The somatic symptoms of military rescuers were apparently higher than their mental symptoms. Mental and pharmaceutical intervention showed good effect on them. Conclusion There is an apparent effect for military rescuers injured seriously psychosomatic health through medication or mental intervention.

Amino acid composition and classification of acidic and alkaline enzymes / 生物工程学报

It is of theoretical and practical significance to understand the mechanism of enzyme adaptation to acidic and alkaline environments and classification of them based on sequence information. In present work, the amino acid composition of 105 acidic and 111 alkaline enzyme sequences was systematically analyzed. Acidic enzymes contained significantly more Trp, Tyr, Thr and Ser, whereas less Glu, Lys, Met and Arg. On the other hand, alkaline enzymes have slightly more Trp, Ala and Cys, whereas less Lys, Arg and Glu. The amount of Ala, Glu, Leu, Asn, Arg, Ser and Thr in acidic and alkaline enzymes varied largely. Hence, a weighted amino acid composition method was developed for the discrimination of acidic and alkaline enzymes. Using the back-check and the 5-fold cross validation methods, the overall accuracy could reach 86.1% and 83.3%, respectively. A new method to classify acidic and alkaline enzymes based on their sequences was established.

Discriminating acidic, neutral and alkaline enzymes with secondary structure amino acid composition / 生物工程学报

In this work, we systematically analyzed the secondary structure amino acid compositions of acidic and alkaline enzymes and compared them with neutral ones. We found that the propensity of the individual residues to participate in secondary structures and the consistently higher composition of neutral and tiny residues might be the general stability mechanisms for their adaptation to pH extremes. Based on this, we presented a secondary structure amino acid composition method for extracting useful features from sequence. The overall prediction accuracy evaluated by the 10-fold cross-validation reached 80.3%. Comparing our method with other feature extraction methods, the improvement of the overall prediction accuracy ranged from 9.4% to 18.7%. The random forests algorithm also outperformed other machine learning techniques with an improvement ranging from 2.7% to 21.8%.

Modeling relationship between sequence characteristics of insecticidal crystal proteins and their inhibitory against Plutella xylostella / 生物工程学报

The knowledge of the relationship between sequence characteristics of insecticidal crystal proteins (ICP) and their inhibitory against Plutella xylostella provided helpful information for the rational design of ICP with desirable activity against Plutella xylostella. The four key loops of ICP with determined activities against Plutella xylostella were selected to study the quantitative relationship between sequence characteristics and insecticidal activity. The first principle components' score vectors for 20 amino acids were assigned to converting amino acids into data. The six key sites X3, X9, X12, X13, X14 and X19 were predicted by stepwise regression method. The amino acids L/ X3, S/ X9, S/ X12, T/ X13, A/ X14 and G/ X19 found by partial least squares regression and second order polynomial models were predicted to increase the activity of ICP against Plutella xylostella.

Prediction of lipases types by different scale pseudo-amino acid composition / 生物工程学报

Lipases are widely used enzymes in biotechnology. Although they catalyze the same reaction, their sequences vary. Therefore, it is highly desired to develop a fast and reliable method to identify the types of lipases according to their sequences, or even just to confirm whether they are lipases or not. By proposing two scales based pseudo amino acid composition approaches to extract the features of the sequences, a powerful predictor based on k-nearest neighbor was introduced to address the problems. The overall success rates thus obtained by the 10-fold cross-validation test were shown as below: for predicting lipases and nonlipase, the success rates were 92.8%, 91.4% and 91.3%, respectively. For lipase types, the success rates were 92.3%, 90.3% and 89.7%, respectively. Among them, the Z scales based pseudo amino acid composition was the best, T scales was the second. They outperformed significantly than 6 other frequently used sequence feature extraction methods. The high success rates yielded for such a stringent dataset indicate predicting the types of lipases is feasible and the different scales pseudo amino acid composition might be a useful tool for extracting the features of protein sequences, or at lease can play a complementary role to many of the other existing approaches.

Random forest for classification of thermophilic and psychrophilic proteins based on amino acid composition distribution / 生物工程学报

We used amino acid composition distribution (AACD) to discriminate thermophilic and psychrophilic proteins. We used 10-fold cross-validation and independent testing with other dataset to evaluate the models. The results showed that when the segment was 1, the overall accuracy reached 92.9% and 90.2%, respectively. The AACD method improved the prediction accuracy when support vector machine was used as the classifier. When six new features were introduced, the overall accuracy of random forest improved to 93.2% and 92.2%, the areas under the receiver operation characteristic curve were 0.9771 and 0.9696, which was better than other ensemble classifiers and comparable with that of SVM.

Predicting the cofactors of oxidoreductases by the modified pseudo-amino acid composition / 生物工程学报

Types of cofactor independency for newly found oxidoreductases sequences are usually determined by experimental analysis. These experimental methods are both time-consuming and costly. With the explosion of oxidoreductases sequences entering into the databanks, it is highly desirable to explore the feasibility of selectively classifying newly found oxidoreductases into their respective cofactor independency classes by means of an automated method. In this study, we proposed a modified Chou's pseudo-amino acid composition method to extract features from sequences and the k-nearest neighbor was used as the classifier, and the results were very encouraging. When lambda = 48, w = 0.1, the areas under the ROC curve of k-nearest neighbor in 10-fold cross-validation was 0.9536; and the success rate was 92.0%, which was 3.5% higher than that of pseudo-amino acid composition. It was also better than all the other 7 feature extraction methods. Our results showed that predicting the cofactors of oxidoreductases was feasible and the modified pseudo-amino acid composition method may be a useful method for extracting features from protein sequences.

Psychological obstacle in peacekeeping officers and soldiers and intervention study / 解放军医学杂志

Peacekeeping is a military operation under certain conditions.In the special operations,the military personnel will have a special emotional response,mainly tension.It includes physical and psychological stress,based on the cognition and evaluation of the environment and forms of military activities.The psychological problems of peacekeepers caused by peacekeeping operation have been identified as one of the major reasons for non-combat attrition.Thus,researches on mental health of peacekeepers should not be ignored.So,we reviewed the interaction between peacekeeping operations and peacekeepers'mental health,and a few psychological intervention techniques to provide theoretical and practical basis for serving peacekeepers with mental health.