ABSTRACT
We constructed bicistronic expression system containing AH6 promoter, 5' UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃, respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4-11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.
Subject(s)
Corynebacterium glutamicum , Promoter Regions, Genetic , Protein Sorting Signals , Protein TransportABSTRACT
We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.
Subject(s)
Bacillus , Glycoside Hydrolases , Metabolism , Molecular Weight , Protein Conformation , Sequence Deletion , Substrate Specificity , TemperatureABSTRACT
Objective To explore the prognosis and management of atrial fibrillation (AF) in patients with atrial septal defect(ASD) accompanied by AF after transcatheter closure of ASD. Methods During the period from July 2010 to May 2013, a total of 24 patients with ASD accompanied by AF were admitted to authors’ hospital to receive transcatheter closure of ASD. Electrocardiogram (ECG), chest X-ray film and transthoracic echocardiography (TTE) were performed before and one day after the operation. Follow-up information was obtained through telephone or at out-patient clinic interview. Results Successful occlusion of ASD was obtained in all patients, and in no patient the AF rhythm turned to sinus rhythm after the procedure. In one patient preoperative AF turned to postoperative atrial flutter, and AF recurred in one case who had received transcatheter ablation of AF before the procedure. One female patient developed gastric bleeding during the course of orally taking warfarin, and she died of cerebral infarction at three days after ceasing the use of warfarin. Of the 24 patients, no anticoagulant drug was used in 5 (20.8%), oral administration of aspirin was given in 7 (29.2%), and oral medication of warfarin was employed only in 11 (45.8%). Conclusion The spontaneous conversion rate of AF is very low in patients with ASD complicated by AF after transcatheter closure of ASD. Postoperative medication of anticoagulation should be strictly standardized and carefully managed.
ABSTRACT
Objective To explore the application value of 64-slice spiral CT cardiac enhancement scanning in the morphological as-sessment of left atrial appendage(LAA).Methods Patients were selected retrospectively between April 16,2013 and May 1,2013, including 142 patients with sinus rhythms underwent MDCT coronary angiography,25 patients with atrial fibrillation underwent MDCT pulmonary vein-left atrial angiography.All images were post-processed,datas were measured and the morphology of left at-rial appendage were classified into different types.Results 167 patients were enrolled,including 88 males and 79 femals,age 53.7± 8.9 years.Anatomical relationship of the orifice of left atrial appendage to left superior pulmonary vein(LSPV)were classified as high type (superior to LSPV,2.4%),middle type(parallel to LSPV,86.2%),low type(inferior to LSPV,11.4%).LAA orifice could be classified into three types including oval(162,97%),round-like(3,1.8%)and drop-like(2,1.2%).LAA morphology could be classified in four types including Cauliflower(35,21%),Windsock(41,24.6%),Cactus(7,4.2%),Chicken-wing(84, 50.3%).Patients with atrial fibrillation have a larger LAA volume than who with sinus rhythm.Conclusion 64-slice spiral CT car-diac enhancement scanning can provide a vivid and intuitive reference to the anatomy of LAA and its adjacent structures,and it can also provide quantitative evaluation of LAA anatomical parameters,so it may have a nice significance in the direction of LAA occlu-sion.