Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp / 生物工程学报

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-β-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 ℃. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 ℃ and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 ℃ and pH 7.0-9.0 and Co2+ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.

Advances in methods for detecting plastics biodegradation / 生物工程学报

The pollution caused by improper handling of plastics has become a global challenge. In addition to recycling plastics and using biodegradable plastics, an alternative solution is to seek efficient methods for degrading plastics. Among them, the methods of using biodegradable enzymes or microorganisms to treat plastics have attracted increasing attention because of its advantages of mild conditions and no secondary environmental pollution. Developing highly efficient depolymerizing microorganisms/enzymes is the core for plastics biodegradation. However, the current analysis and detection methods cannot meet the requirements for screening efficient plastics biodegraders. It is thus of great significance to develop rapid and accurate analysis methods for screening biodegraders and evaluating biodegradation efficiency. This review summarizes the recent application of various commonly used analytical techniques in plastics biodegradation, including high performance liquid chromatography, infrared spectroscopy, gel permeation chromatography, and determination of zone of clearance, with fluorescence analysis techniques highlighted. This review may facilitate standardizing the characterization and analysis of plastics biodegradation process and developing more efficient methods for screening plastics biodegraders.

Enzyme production mechanism of anaerobic fungus Orpinomyces sp. YF3 in yak rumen induced by different carbon source / 生物工程学报

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.

Prokaryotic expression and activity detection of insulin-degrading enzyme mutant T142A / 中国生物制品学杂志

@#Objective To express insulin-degrading enzyme(IDE)mutant T142A in prokaryotic cells and detect its activity.Methods According to the results of multi-sequence alignment and IDE substrate co-crystal structure,an active residue in β6-strand structure of IDE were predicted.The recombinant plasmid ppSUMO-T142A,with the site mutation of threonine 142 to alanine,was constructed by point mutation technique and expressed by E.coli prokaryotic expression system.After purification by nickel ion column affinity chromatography,ion exchange chromatography and gel filtration chromatography,the mutant T142A was obtained and determined for the activity by fluorescence method.Results IDE amino acid sequence is highly conserved among 16 species.T142 directly participates in substrate binding,interacts with substrate cleavage sites,and is close to important structures such as catalytic active sites and door-subdomains.The mutation of recombinant plasmid ppSUMO-T142A was proved to be correct by sequencing.The expressed fusion protein His-SUMO-T142A was mainly existed in soluble form in the supernatant at a concentration of 18 mg/mL,with a relative molecular mass of about 131 000;After three steps of purification,the purity of mutant T142A reached 86%.The maximum reaction rate(V_(max))of T142A catalytic degradation of fluorescent substrate V was 501.06 min~(-1) and the Michaelis constant(K_m) was 9.01μmol/L.Compared with wild-type IDE(V_(max) was 2 814.32 min~(-1),K_m was 11.93μmol/L),the activity of T142A decreased significantly.Conclusion The activity of IDE mutant T142A expressed in this study greatly decreases,while T142 is an important residue for IDE to play its enzymatic function,which provides an experimental basis for the development of new IDE activity regulatory molecules.

Research progress of novel drug therapy for antibody-mediated rejection in kidney transplantation / 中华泌尿外科杂志

Antibody-mediated rejection (AMR) is the primary factor affecting the long-term prognosis of kidney transplant recipients and kidney allograft. Currently, there is no universally recognized or approved drug for the treatment of AMR. Therefore, more novel drug studies and clinical trials are urgently needed in order to change the long-term prognosis of kidney transplant recipients. Based on the core principles of prevention and treatment of AMR, this paper discusses the mechanism and efficacy of several new types of drugs of most concern in the treatment of AMR from three aspects: removing donor specific antibody, blocking antibody-mediated and complement-mediated tissue damage, and inhibiting the proliferation and activation of antibody-producing cells. These emerging drugs have shown potential in preventing and treating AMR and improving the prognosis of recipients, which is expected to change the dilemma of AMR treatment in the future and provide more effective treatment options for improving the long-term prognosis of kidney transplant recipients.

Expression, purification, and functional identification of immunoglobulin degrading enzyme IdeS in Escherichia coli / 药学学报

In the process of evolution, pathogenic Streptococcus pyogenes secretes an immunoglobulin G-degrading enzyme IdeS which can specifically cleave the hinge region of immunoglobulin G in order to escape the immune response against the host. On the one hand, IdeS can be used for IgG fingerprinting as a tool enzyme combined with mass spectrometry technology. On the other hand, IdeS can be used to treat the antibody-responsive diseases produced by autoimmunity as a therapeutic protein. In this study, the backbone of plasmid pCold was used to construct two expression vectors of recombinant protein IdeS, which were heterologously expressed in Escherichia coli Shuffle T7. After purification by affinity chromatography, the recombinant IdeS activity was detected and their activity differences between the two were compared. Among them, the yield of the recombinant IdeS containing the His6-tag at the N-terminus was 4 mg·L-1, and the cleavage reaction with antibody IgG1 at 1∶200 (m/m) at 37 ℃ for 30 min could complete. However, the yield of the recombinant IdeS containing both the N-terminal His6 tag and the C-terminal silica affinity tag (silica bing peptide, SiBP) is 1.5 mg·L-1, and the degradation reaction with antibody IgG1 at 1∶20 (m/m) at 37 ℃ for 30 min could reach the end. The C-terminal fusion peptide has a great influence on the yield and activity of IdeS, which is not conducive to subsequent application in drug development. Above all, the recombinant IdeS containing the His6-tag at the N-terminus expressed by this system has high activity and can fully meet the needs of antibody drug development and mapping analysis of IgG.

Research progress on desensitization therapy for highly sensitized kidney transplant recipients / 器官移植

Human leukocyte antigen (HLA) sensitization has been previously considered as a contraindication for kidney transplantation. In the past 30 years, with the development of desensitization therapy strategies and immunosuppressants, more and more highly sensitized patients have been eligible for kidney transplantation. However, highly sensitized patients still face a high incidence of hyperacute rejection and antibody-mediated rejection following kidney transplantation, which restricts the success of kidney transplantation and long-term graft survival. At present, exploring effective desensitization regimen is a hot spot in the research of organ transplantation. In this article, the current desensitization therapy strategies, new preparations for desensitization therapy, and the benefits and risks of desensitization therapy were reviewed, and the current status and future direction of desensitization therapies were investigated, aiming to provide reference for resolving the immune barrier, improving the success rate of kidney transplantation and enhancing the quality of life of highly sensitized recipients.

Development of biodegradation process for Poly(DL-lactic acid) degradation by crude enzyme produced by Actinomadura keratinilytica strain T16-1

Background: Plastic waste is a serious problem because it is difficult to degrade, thereby leading to global environment problems. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester derived from renewable resources, and it can be degraded by various enzymes produced by microorganisms. This study focused on the scale-up and evaluated the bioprocess of PLA degradation by a crude microbial enzyme produced by Actinomadura keratinilytica strain T16-1 in a 5 L stirred tank bioreactor. Results: PLA degradation after 72 h in a 5 L bioreactor by using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. However, the PLA degradation process inadvertently produced lactic acid as a potential inhibitor, as shown in our experiments at various concentrations of lactic acid. Therefore, the dialysis method was performed to reduce the concentration of lactic acid. The experiment with a dialysis bag achieved PLA degradation by weight loss of 99.93%, whereas the one without dialysis achieved a degradation of less than approximately 14.75%. Therefore, the dialysis method was applied to degrade a commercial PLA material (tray) with a conversion efficiency of 32%, which was 6-fold more than that without dialysis. Conclusions: This is the first report demonstrating the scale-up of PLA degradation in a 5 L bioreactor and evaluating a potential method for enhancing PLA degradation efficiency.

Effect of Hei Xiaoyaosan on Expressions of Aβ1-42, GSK-3β, NEP, IDE in Hippocampus Area of Alzheimer's Dementia Mice / 中国实验方剂学杂志

Objective: To observe the effect of Hei Xiaoyaosan on expressions of β-amyloid 1-42 peptide(Aβ1-42),glycogen synthase kinase-3β(GSK-3β),neprilysin(NEP),insulin-degrading enzyme(IDE) in the hippocampus area of Alzheimer's dementia mice. Method: After weighing, 42 APP/PSI bivalent transgenic mice were randomly divided into 4 groups:10 mice in the model group, 10 mice in the positive drug control group, 11 mice in the high-dose Hei Xiaoyaosan group, and 11 mice in the low-dose Hei Xiaoyaosan group; 10 wild C57BL/6J mice of the same age and strain were used for negative control group. Drugs were administered to mice by gavage once a day for 12 weeks. Then the behavior of all the mice were detected by Morris water maze, the morphological changes in hippocampal neurons were observed by hematoxylineosin(HE) staining, the expressions of Aβ1-42, GSK-3β, NEP and IDE proteins in hippocampus were detected by immunohistochemistry. Result: After 3 months of treatment, compared with negative control groups, the average escaping latency periods prolonged significantly, and the number of cross-platform was decreased significantly in model group (Pβ1-42 and GSK-3β proteins in model mice hippocampus were significantly increased (PPPβ1-42 and GSK-3β proteins in the hippocampus of drug groups were significantly decreased (PPPConclusion: Hei Xiaoyaosan can significantly improve the learning and memory abilities of AD mice, which may be related to the reduction of cognitive impairment in AD mice by regulating abnormal deposition and degradating Aβ in the hippocampus.

Kidney-reinforcing and Governor Vessel-regulating EA Intervention May Improve Learningmemory Possibly by Suppressing Formation of Senile Plaques in Hippocampus in APP/PS 1 Double Transgenic Alzheimer's Disease Mice / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on learning-memory ability and the expression of senile plaques (SP), amyloid precursor protein (APP), β-secretase 1(BACE 1) and insulin degrading enzyme (IDE) in the hippocampus in APP/presenilin 1 (PS 1) double transgenic Alzheimer's disease (AD) mice, so as to reveal its mechanisms underlying improvement of AD. METHODS: A total of 18 male APP/PS 1 double transgenic AD mice were randomly divided into model, EA-2-week and EA-3-week groups (n=6 in each). The control group was consisted of 6 male wild mice. EA (2 Hz, 2 mA) was applied to "Baihui" (GV 20) and bilateral "Shenshu" (BL 23) for 15 min, once a day, with 7 days being a therapeutic course, 2 or 3 courses altogether and with an one day's interval between every two courses. The spatial learning-memory ability was assessed using Morris water maze test during 5 days' training. The immunoactivity of SP in the hippocampus tissue was detected by immunohistochemistry, and the expression levels of APP, BACE 1 and IDE in the hippocampus were analyzed by Western blot. RESULTS: Following modeling, the escape latency and path length of hidden platform tests were significantly increased (P<0.01, P<0.05), and the platform crossing time of spatial probing test significantly decreased (P<0.01) in the model group compared with the control group. After EA intervention, the escape latency on the 5th day of training, and the path length on the 4th and 5th day of training in both EA-2-week and EA-3-week groups were significantly shorter relevant to the model group (P<0.01), and those of the EA-3-week group were considerably shorter than those of the EA-2-week group in the escape latency and path length (P<0.05, P<0.01). The platform crossing times of spatial probing test were significanthy increased in both EA-2-week and EA-3-week groups in comparison with the model group (P<0.01), and that of the EA-3-week group was considerably increased compared with the EA-2-week group (P<0.05). Immunohistochemical staining showed that the number of SP in the hippocampus was markedly increased in the model group compared with the control group (P<0.01), and was markedly reduced in both EA-2-week and EA-3-week groups (P<0.01), and that of the EA-3-week group was significantly decreased compared with the EA-2-week group (P<0.01). The expression levels of hippocampal APP and BACE 1 proteins were significantly higher in the model group than in the control group (P<0.01), and that of hippocampal IDE was markedly lower in the model group than in the control group (P<0.01). After EA, the increased expression levels of APP and BACE 1 proteins and the decreased expression level of IDE in the EA-2-week and EA-3-week groups were significantly inhibited (P<0.01). The effects of EA-3-week were significantly stronger than those of EA-2-week in down-regulating the expression of APP and BACE 1 proteins and up-regulating the expression of IDE (P<0.01, P<0.05). CONCLUSION: EA stimulation of GV 20 and BL 23 can improve the learning-memory ability in APP/PS 1 double transgenic AD mice, which may be related to its effects in down-regulating the expression of SP, APP and BACE 1 proteins and up-regulating the expression of IDE protein in the hippocampus.

Purification, characterization and partial primary structure analysis of rutin-degrading enzyme in tartary buckwheat seeds / 生物工程学报

Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²⁺, Zn²⁺, Mn²⁺ and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.

Phytase-Producing Bacteria from Extreme Regions in Indonesia

ABSTRACTIn this study, 154 isolates capable of producing extracellular phytate-degrading activity were isolated from four soil samples from volcanic areas in Central Java, Indonesia. Six strains with high phytate-degrading activity were selected for strain identification and characterization of the corresponding phytate-degrading enzyme. Blast analysis of 16S rRNA gene sequences revealed high similarities for all the six isolates to reference sequences belonging to the genusBacillus. Isolates MS5, MC6, D10 and D16 showed 99% sequence identity toB. cereus, while isolate MC8 exhibited 99% sequence identity toB. aryabhatti and D6 99% sequence identity toB. psychrotolerans. The crude extracellular phytase preparations from the isolates showed following optimal conditions for phytate dephosphorylation: pH 4.0 and 50°C (isolate D10), pH 5.0 and 60°C (isolate MC6, and isolate MS5), pH 6.0 and 50°C (isolate D16) and pH 6.0 and 60°C (isolate D6) and pH 6.0 and 40°C (isolate MC8). Zn2+ and Fe3+ strongly inhibited phytate dephosphorylation with all phytase preparations studied. In the presence of Ca2+, an increase in phytase activity of 10-15% was obtained.

Influence of anesthesia and surgery on the expression of transport receptors and catabolic enzymes of amyloid β-protein in aged rats / 解放军医学杂志

Objective: To investigate the expression changes in transport receptor and catabolic enzymes of amyloid β-protein (Aβ) in the brain of aged rats after surgery.

Regulation of Zi-Bu Pi-Yin Method on β-amyloid Peptide and Insulin Degrading Enzyme in Brain Tissues of Spleen Yin Deficiency Diabetic Rats / 世界科学技术-中医药现代化

This study was aimed to observe different forms of β-amyloid peptide (Aβ) and insulin degrading enzyme (IDE) in the hippocampus and cortex in order to further explore the role of Aβ and IDE on spleen yin deficiency di-abetes-associated cognitive decline (DACD), and the effect of Zi-Bu Pi-Y in method. The rats were randomly divided into five groups, which were the blank control (Cont) group, diabetes (DM) group, spleen yin deficiency (pi) group, spleen yin deficiency diabetes (piDM) group and Zi-Bu Pi-Y in recipe (ZBPYR) group. Soluble and insoluble Aβ in the hippocampus and cortex of rats were extracted by gradient centrifugation, and then measured by ELISA. The ex-pression of IDE was observed by western blot. The results showed that the content of soluble and insoluble Aβ1-42 in the hippocampus and cortex of the DM group and piDM group were higher than the Cont group. The soluble and in-soluble Aβ1-42 content in the hippocampus and cortex of the ZBPYR group were reduced compared with the DM group and the piDM group. The soluble Aβ1-40 in the cortex of the DM group, pi group and piDM group were in-creased compared with the Cont group (P < 0.05). The soluble Aβ1-40 content of the ZBPYR group was decreased compared with the DM group and the piDM group (P < 0.05). The expression of IDE protein was decreased in the hippocampus of the DM group and the piDM group compared with the Cont group (P< 0.05), and the IDE protein level in the hippocampus of the ZBPYR group was increased compared with the DM group and the piDM group (P<0.05). The expression of IDE protein in the cortex of the DM group, pi group and piDM group was lower than the Cont group (P< 0.05). The IDE protein level in the cortex of the ZBPYR group was reduced compared to the DM group (P< 0.05). It was concluded that the increased Aβ1-42 in brain may be a major pathological change of DACD and spleen yin deficiency DACD. The decreased IDE expression may be one of the reasons to induce increasing of Aβ1-42 level. The Zi-Bu Pi-Y in method may decrease the Aβ1-42 content by upregulating IDE protein expression.

The effects of rehabilitation training on amyloid-beta peptide and insulin-degrading enzyme levels in the hippocampus of rats with vascular dementia / 中华物理医学与康复杂志

Objective To investigate the effects of rehabilitation training on hippocampal amyloid-beta peptide (Aβ) and insulin-degrading enzyme (IDE) levels in vascular dementia (VD).Methods Thirty female Sprague-Dawley rats were randomly assigned to a rehabilitation group (n =10),a model group (n =10) or a sham-operation group (n =10).An experimental VD model was established in the rats of the first 2 groups by bilateral common carotid artery permanent ligation.The rats in the rehabilitation group then received 1 h of rehabilitation training daily.Learning and memory were assessed at 4 weeks aftet the operation.Immunohistochemical staining was used to detect Aβ and IDE expression in the hippocampus dentate gyrus (DG) area.Results The rats in the rehabilitation group showed significantly better learning ability compared with the model group.The expression of Aβ in the rehabilitation group was significantly less than in the model group.The expression of IDE in the rehabilitation group was significantly greater Conclusion Rehabilitation can accelerate the recovery of learning and memory in VD,at least in rats The mechanism is possibly related to decreased accumulation of Aβ in the hippocampus due to up-regulation of the expression of IDE.

The study on the role of transcription factor GATA binding protein 3 in familial Alzheimer's disease pathogenesis / 中华神经科杂志

Objective To investigate the mechanisms of decreasing insulin degrading enzyme (IDE) level by mutation V97L in the gene presenilin 1 (PS-1).Methods Transcription factor GATA binding protein 3 (GATA-3) activity was assessed by protein/DNA array and verified by Western blot in SH-SY5Y cells transfected by PS-1 mutation V97L.Results Protein/DNA array and Western blot revealed that there was increased transcript factor activity (5.5 times high) and protein level of GATA-3 in V97L-PS-1 transfected SH-SY5Y cells.Transcription factor GATA-3 can bind to the IDE promoter and negatively control the IDE transcription level.Conclusion PS-1 mutation V97L may regulate the transcription of IDE via GATA-3, and subsequently involve in deposition of Aβ42 and development of Alzheimer's disease.

Overexpression of Insulin Degrading Enzyme could Greatly Contribute to Insulin Down-regulation Induced by Short-Term Swimming Exercise / 한국실험동물학회지

Exercise training is highly correlated with the reduced glucose-stimulated insulin secretion (GSIS), although it enhanced insulin sensitivity, glucose uptake and glucose transporter expression to reduce severity of diabetic symptoms. This study investigated the impact of short-term swimming exercise on insulin regulation in the Goto-Kakizaki (GK) rat as a non-obese model of non-insulin-dependent diabetes mellitus. Wistar (W/S) and GK rats were trained 2 hours daily with the swimming exercise for 4 weeks, and then the changes in the metabolism of insulin and glucose were assessed. Body weight was markedly decreased in the exercised GK rats compare to their non-exercised counterpart, while W/S rats did not show any exercise-related changes. Glucose concentration was not changed by exercise, although impaired glucose tolerance was improved in GK rats 120 min after glucose injection. However, insulin concentration was decreased by swimming exercise as in the decrease of GSIS after running exercise. To identify the other cause for exercise-induced insulin down-regulation, the changes in the levels of key factors involved in insulin production (C-peptide) and clearance (insulin-degrading enzyme; IDE) were measured in W/S and GK rats. The C-peptide level was maintained while IDE expression increased markedly. Therefore, these results showed that insulin down-regulation induced by short-term swimming exercise likely attributes to enhanced insulin clearance via IDE over-expression than by altered insulin production.

Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3 percent referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55ºC. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K M = 54 µmol l-1 and k cat = 190 sec-1 at pH 5.0 and 37ºC. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

Regulation of ? amyloid protein level in the brain / 中国临床药理学与治疗学

? amyloid protein(A?) including A?40 and A?42 are the important bioactive substances in vivo.Their toxic and beneficial attributes in the body depend on its concentration.The brain A? level is maintained by two balances under the physiological condition.The first balance is the generation involved in ?-secretase and ?-secretase and the degradation involved in neprilysin(NEP) and insulin-degrading enzyme(IDE) of A?. The second one is the balance between the receptor for advanced end glycation products(RAGE)-mediated influx and low-density lipoprotein receptor related protein 1(LRP1)-mediated efflux of A? across the blood-brain barrier(BBB).Breakdowning any one of the two balances would result in the aggregation and precipitation of A? in the brain,which is a crucial event in the pathogenesis of Alzheimer's disease(AD).This paper reviews the regulation of brain A? level under the physiological condition and the reducing strategies on the level of brain A? under the pathological condition for developing new drugs in the treatment of AD.

Heterogenous Gene Expression of Methyl Parathion Hydrolase and Analysis of the Enzyme Activity / 微生物学通报

Methyl parathion hydrolase (MPH, E.C.3.1.8.1) coding gene mph from Pseudomonas sp. WBC-3, isolated and identified by our lab, was successfully expressed in E. coli AD494 (DE3)/ pET32a(+) system as soluble fusion form at high level. The recombinant MPH showed nearly 4～5 fold higher specific activity to parathion than the enzyme from Pseudomonas sp. WBC-3. In addition, the thermal stability of the recombinant enzyme was improved comparing with the wild type enzyme.