Hemicellulose is a kind of very abundant carbohydrate,which is not y ot exploited Arabinofuranosidase is important enzyme in biodegradation of hemi cellulose Up to now many arabinofuranosidases and genes have been studied in t he world In this paper, we reviewed mainly on the classification, characterizati on, utility and gene expression of arabinofuranosidase

In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.
Biotechnology ; Cold Temperature ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Plasmids ; genetics ; Recombinant Proteins

The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; L-Lactate Dehydrogenase ; biosynthesis ; Molecular Weight ; Recombinant Proteins ; biosynthesis ; Temperature ; Thermotoga maritima ; enzymology

The knock out vector pHK was constructed with E coli vector pET 28a and shuttle vector pHY300PL, by using denatured DNA and homologous recombination technique, the kanamycin resistance gene ( Kan r) from integrated alkaline protease gene engineering strain BP071 was knocked out successfully, and the 11 positive clones were obtained The yield of the best positive clone BP0715 was stable as same as BP071 The methods provided the good experience for the industrial microbiology research, and it was foundation for studying on the safety of genetically modified organisms

Thermophiles can produce cellulosic ethanol at a high temperature where ethanol is directly distillated from fermentation, and biodegradation of lignocellulose can be simultaneously achieved when these thermophiles carry and express cellulase and hemicellulase genes. The simultaneous biodegradation, fermentation and distillation, a three-in-one process, can result in low production costs of cellulosic ethanol. We reviewed the advances and challenges in the approach to the three-in-one process, which refer to lignocellulases, regulation mechanisms, and genetic transfer systems.
Bacteria ; genetics ; metabolism ; Cellulose ; metabolism ; Ethanol ; metabolism ; Fermentation ; Hot Temperature ; Thermoanaerobacterium ; metabolism

The gene of beta-glucuronidase from Thermotoga maritima was cloned into the plasmid pHsh, and expressed in Escherichia coli JM109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 65.9 kD. The optimal activity of beta-glucuronidase was found at pH 5.0 and 80 degrees C. The purified enzyme was stable over a pH range from 5.8 to 8.2 and had a half life of 2 h at 80 degrees C. The kinetic experiments at 80 degrees C with p-nitrophenyl-beta- glucuronide as substrate gave a K(m) and V(max) of 0.18 mmol/L and 312 u per mg of protein. The purified enzyme could transform glycyrrhizin to glycyrrhetinic acid.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; enzymology ; genetics ; Glucuronidase ; biosynthesis ; genetics ; metabolism ; Glycyrrhetinic Acid ; metabolism ; Glycyrrhizic Acid ; metabolism ; Hot Temperature ; Kinetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Thermotoga maritima ; enzymology ; genetics

The structure gene pelA from Thermotoga maritima MSB8 encoding pectate lyase was amplified and ligated into pHsh, resulting pHsh-pelA. Through structural optimization on pHsh-pelA, the ultimate plasmid, pHsh-pelC, which possessed the most appropriate structure and free energy of mRNA, was obtained. Pectate lyase C (PelC) was obtained after expressing pHsh-pelC in Escherichia coli JM109. The optimum activity of PelC was determined at pH 8.5 at 90 degrees C, with a half-life for almost 2 h at 95 degrees C. PelC was stable at the pH range of 8.2-9.8, and was dependent on Ca2+ for activity and stability. The enzyme kept stable for a long time and possessed a high level of activity at 60 degrees C. The kinetic assay using polygalacturonic acid (PGA) as substrate gave K(m) and V(max) of 0.11 mmol/L and 327 U per mg of protein. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant PelC was about 43 kD, which was exactly the size predicted. The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and cheap induction. Moreover, the superior stability of the recombinant enzyme laid the base for large-scale fermentation application.
Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Hot Temperature ; Polysaccharide-Lyases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombination, Genetic ; Thermotoga maritima ; enzymology

Objective:To study the physical activity level and its influence factors among residents in one suburb of Beijing,so as to provide specific interventions for different people in different circum-stances and to provide reference for health relevant policy-making in the future.Methods:In the study, 7 31 9 subjects aged 1 8 years or above were involved.The self-designed questionnaires based on Health Belief Model (HBM)had acceptable validity and reliability.The physical activity levels were calculated to classify sufficient or insufficient amount by a thousand-step equivalent greater than or equal to 6 or 1 0. Multiple variable Logistic regression was used to explore the influence factors of the physical activity among the residents.Results:The residents’median amount of physical activity in the suburb district of Beijing were 9.1 thousand-step equivalent with quartile of (3.8,20.4).The percentages of the thou-sand-step equivalent greater than or equal to 6 or 1 0 were 63.7% and 47.7%,respectively.The median amounts of physical activity from work or household chores,transportation and recreation physical activi-ties were 4.0,1 .0,0.0 and the components of the total amount of physical activity from those were 61 .7%,1 8.3% and 20.1 %,respectively.There were 8.6% residents whose life did notinvolve moder-ate or vigorous intensity activities.By using factor analysis,five factors were extracted from the scale based on the HBM;These factors together contributed to 63.7% of the sum of the squared loadings.The differences of physical activity levels on education level,age,gender,self-efficacy,cues,subjective and objective barriers were statistically significant (P <0.05).Those who were female,with older age,lower education level,higher self-efficacy,fewer cues,fewer subjective and objective barriers preferred to do more physical activities.Conclusion:The physical activity levels among the residents in the suburb dis-trict of Beijing are moderate and high,and most amount of physical activities from work or household chores.Those who are male and whose ages are from 1 8 to 29 years and whose education levels are of university or above should be focused on intervention.Specific interventions should be developed for dif-ferent people in different situations;More attention should be paid to improve the residents’self-efficacy and reduce the subjective and objective barriers of physical activity,and we also should actively advocate people to have more leisure exercise so as to improve the physical activity level among all residents.

AIM: To investigate the effect of extracorporeal membrane oxygenation (ECMO) on the pharmacokinetics/pharmacodynamics (PK/PD) of daptomycin in critically ill patients. METHODS: Twenty four patients with severe infection in our hospital were randomly selected and divided into ECMO group and non ECMO group. They were intravenously injected with daptomycin 500 mg qd. After the drug reached the stability statement, venous blood was collected at different time points before and after the infusion. The plasma drug concentration was measured and the pharmacokinetic parameters were calculated. The probability target acquisition (PTA) and the cumulative fraction response (CFR) were calculated by Monte Carlo simulation. RESULTS: After dosing, the main pharmacokinetic parameters in ECMO and non-ECMO group were calculated and listed as follows: C