Biomedical subjects

Bin Yao

Publications and source records attributed to Bin Yao.

At least 19 recordsLinked to original sources

PubMed2007

Expression of xylanase with high specific activity from Streptomyces olivaceoviridis A1 in transgenic potato plants (Solanum tuberosum L.).

The gene, xynB, from Streptomyces olivaceoviridis A1 encoding xylanase, XYNB, with a high specific activity for xylan, was transformed into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens. The integration of xynB into genomic DNA was confirmed by PCR and reverse transcriptase-PCR. The gene was expressed under the control of a constitutive double cauliflower mosaic virus (CaMV) 35S promoter. Both SDS-PAGE and western blot analysis showed high levels of expression of the 21 kDa and 31 kDa XYNB proteins in transgenic potato plants transformed by the binary vectors pBinXy and signal peptide contained pBinSPXy, respectively. The recombinant XYNB protein was present at up to 5% of total soluble leaf protein in the cytoplasm. In transgenic leaf and tuber extracts, xylanase activity was up to 87 micromol min(-1) g(-1) fresh leaf (9.7 micromol min(-1) mg(-1) total soluble protein). The xylanase was stable at 60 degrees C and 70 degrees C in buffers (pH 5.2) for 5 min. Furthermore, the xylanase enzymatic activity remained virtually unchanged over several generations of potato. These results demonstrate that the transgenic potato can be used to produce recombinant xylanase with high specific enzyme activity and can potentially be an alternative to present-day xylanase additives to animal feed.

Endo-1,4-beta Xylanases↗

PubMed2006

A novel phytase with preferable characteristics from Yersinia intermedia.

A Yersinia intermedia strain producing phytase was isolated from glacier soil. The phytase gene, appA, was isolated by degenerate PCR and TAIL-PCR. The full-length fragment contained 2354bp with a 1326-bp open reading frame encoding 441 amino acids. APPA contained the active site RHGXRXP and HD sequence motifs that are typical of histidine acid phosphatases. To our knowledge, this is the first report of the detection of phytase activity and cloning of the relevant gene from Y. intermedia. The gene was overexpressed in Pichia pastoris, and the purified recombinant APPA had a specific activity for sodium phytate of 3960U/mg, which is higher than that of the Citrobacter braakii phytase (previously the highest specific activity known). Recombinant APPA had high activity from pH 2 to 6 (optimum 4.5) and optimal temperature of 55 degrees C; the enzyme was resistant to pepsin and trypsin. These characteristics suggest that APPA may be highly suitable for use in the feed industry.

6-Phytase↗

PubMed2006

Prophylaxis against carcinogenesis in three kinds of unestablished tumor models via IL12-gene-engineered MSCs.

Mesenchymal stem cells (MSCs) were adenovirally engineered to secrete interleukin-12 (AdIL-12-MSCs) and evaluated for their anticarcinogenesis efficacy against three kinds of unestablished tumor models including B16 melanoma, LLC Lewis lung cancer and HCC hepatoma. Injection of AdIL-12-MSCs into protected mice before tumor inoculation prevented all of 12 mice in B16 preventive groups, 10 out of 12 in LLC lung cancer model and 11 out of 12 mice in HCC hepatoma model from developing tumors, whereas the control groups pre-receiving PBS were validated for 100% carcinogenesis; the tumor formation rates in free-AdIL-12 and vacant MSC groups were unveiled between approximately 83 and 100% even with plentiful angiogenesis and newborn lymphatic vessels, as well as distant metastases. As a novel approach, AdIL-12-MSC has revealed expected preventive effects on carcinogenesis (P<0.01) with low-toxic, broad-spectrum and long-range superiorities. In conclusion, our data indicate that AdIL-12-MSC possess the potential for tropism to preclinical tumor lesions and deprives surviving or hibernating tumor cells, which have escaped from conventional treatments, of revival and recurrence.

Animals↗

PubMed2006

Validation of a patient satisfaction questionnaire for anemia treatment, the PSQ-An.

BACKGROUND: Treating anemia associated with chemotherapy and many cancers is often necessary. However, patient satisfaction with anemia treatment is limited by the lack of validated instruments. We developed and validated a new treatment-specific patient satisfaction instrument: the Patient Satisfaction Questionnaire for Anemia Treatment (PSQ-An). Treatment burden and overall satisfaction scales were designed for ease of use in clinical practice. METHODS: 312 cancer patients (141 breast, 69 gynecological, and 102 non-small cell lung) were targeted to complete the PSQ-An at 4 week intervals. Data from weeks 5 and 9 were analyzed. Patients also completed the MOS SF-36 Global Health assessment and questions concerning resources devoted to anemia treatment. Item reduction used endorsement rates, floor/ceiling effects, and item-item correlations. Factor analysis identified meaningful subscales. Test-retest reliability was assessed. Construct validity was tested, using Pearson's correlations, by comparing subscale scores to Global Health, hemoglobin levels, and resources devoted to anemia treatment. RESULTS: The overall response rate was 92.9% (264/284) at week 5. Most (84.2%) of the patients were female, and the mean (SD) age was 60.2 (+/- 11.8) years. Two distinct subscales were identified measuring treatment burden (7 items) and overall satisfaction (2 items). Test-retest reliability was examined (ICC: 0.45-0.67); both were internally consistent (alpha = 0.83). Both subscales exhibited convergent and divergent validity with independent measures of health. ANOVA results indicated that the PSQ-An Satisfaction subscale discriminated between 5 levels of MOS SF-36 Global Health (P = 0.006). CONCLUSION: The PSQ-An is a validated, treatment-specific instrument for measuring satisfaction with anemia treatment for cancer patients. PSQ-An subscales reflect the burden of injection anemia treatment on cancer patients and their assessment of the overall treatment value.

Aged↗

PubMed2006

Expression of organophosphorus hydrolase OPHC2 in Pichia pastoris: purification and characterization.

Organophosphorus hydrolase is able to hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. In this work, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Pichia pastoris at a high expression level (approximately 5.5 g/L) using 3 L high-cell-density fermentation. The expression level is higher than those produced in other expression systems. The results of the SDS-PAGE and the Western blot analyses showed a major 36 kDa polypeptide band, which was the same size as that from the original bacteria, Pseudomonas pseudoalcaligenes C2-1. The expressed enzyme was recovered from the culture supernatant and purified by a single-step purification procedure with a recovery rate of 72.78%. The main physiochemical features of the recombinant OPHC2, including its optimum temperature and pH for the reaction, its temperature and pH stability, and its sensitivity to some metal ions and chemical reagents, were also characterized. With methyl parathion as a substrate, the optimum temperature and pH for enzyme activity are 65 degrees C and pH 9.0, respectively. It also shows good thermal and pH stability.

Aryldialkylphosphatase↗

PubMed2006

[Improving phytase expression by increasing the gene copy number of appA-m in Pichia pastoris].

In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZalpha-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZalpha is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZalpha to construct the recombinant vector pAOXZalpha-appA-m. The recombinant Pichia pastoris 74#, which already contains one copy of appA-m and its fermentation potency exceeded 7.5 x 10(6) IU/mL, was used as the host strain for the transformation of pAOXZalpha-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supernatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2 x 10(7) IU/mL, which was about 1.6-fold compared with that of the host strain 74#. Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.

6-Phytase↗

PubMed2006

[Improvement of the thermostability of xylanase by N-terminus replacement].

The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with corresponding region of Thermomonosporafusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in Escherichia coli BL21 and Pichia pastoris GS115. TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 degrees C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of TB were all about six-fold of XYNB's after incubating the properly diluted enzyme solutions at 80 degrees C and 90 degrees C for 3min, respectively. The pH stability of TB was 5 to approximately 9, which was narrower than that of XYNB. Still, TB remains a high specific activity as XYNB does. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the discussion for the relationship between structure and function of xylanase was given.

Amino Acid Sequence↗

PubMed2006

[Purification and properties of Citrobacter freundii phytase].

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26) catalyses the stepwise hydrolysis of phytic acid (myo-inositol hexakisphosphate). Phytases are of great commercial importance due to their usage as supplement of food and animal feed, which can cater to nutrition demands and alleviate environmental problems, has been approved by many countries. Although acid phytases have been extensively studied, information regarding the phytases from Citrobacter is limited. In the work presented, a phytase was separated from Citrobacter freundii. After steps of electrophoretic homogeneity by successive ammonium sulfate between 60% and 80% saturation precipitation, DEAE-Sepharose ion-exchange chromatography and gel filtration through Superdex HR 10/30, final gel elution resulted in a 41.3-fold purification and yield of 9.3%. Gel elution is an effective method to purify the protein which contaminated with a few other proteins. The purified preparations were used in subsequent characterization studies. Based on SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 45.0kDa in monomeric form. The pure enzyme has an optimum pH of 4.0 to approximately 4.5. It was found stable between pH5.0 to approximately 7.0, about 90% of the enzyme activity was retained at 37 degrees C for 60min. The phytase has an optimum temperature of 40 degrees C which was lower than that of other phytases from Aspergillus or E. coli (average 50 to approximately 60 degrees C) and was close to the temperature of gastrointestinal tract in animals (37 to approximately 40 degrees C). Thus the enzyme is a promising candidate for animal feed applications. Activity of the purified phytase was influenced by changing the reaction temperature. Data showed that the enzyme retained its activity over a long period when stored at 4 degrees C, whereas thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 60 degrees C for 4min. The Km values of the phytase for dodecasodium phytate at 37 degrees C was 0.85nmol/L with a Vmax 0.53IU/(mg x min). Phytase activity was strongly inhibited by SDS, Zn2+ and moderately inhibited by Cu2+, Cr3+, Fe2+ and Fe3+. Activity was not significantly affected by EDTA, K+, Mg2+ and Ca2+. The phytase has excellent resistance to trypsin, but not pepsin. The N-terminal amino acids sequence of the phytase protein was determined as QCAPEGYQLQQVLMM which exhibited about 80% homology to Glucose-1-phosphatases from E. coli, Shigella flexneri and Salmonella, whereas it did not show apparent sequence similarity with any other phytase listed in the databases. Initial characterization of the purified enzyme suggested that it is a potential candidate for use as an animal feed supplement.

6-Phytase↗

PubMed2005

Knowledge-discovery incorporated evolutionary search for microcalcification detection in breast cancer diagnosis.

OBJECTIVES: The presence of microcalcifications (MCs), clusters of tiny calcium deposits that appear as small bright spots in a mammogram, has been considered as a very important indicator for breast cancer diagnosis. Much research has been performed for developing computer-aided systems for the accurate identification of MCs, however, the computer-based automatic detection of MCs has been shown difficult because of the complicated nature of surrounding of breast tissue, the variation of MCs in shape, orientation, brightness and size. METHODS AND MATERIALS: This paper presents a new approach for the effective detection of MCs by incorporating a knowledge-discovery mechanism in the genetic algorithm (GA). In the proposed approach, called knowledge-discovery incorporated genetic algorithm (KD-GA), the genetic algorithm is used to search for the bright spots in mammogram and a knowledge-discovery mechanism is integrated to improve the performance of the GA. The function of the knowledge-discovery mechanism includes evaluating the possibility of a bright spot being a true MC, and adaptively adjusting the associated fitness values. The adjustment of fitness is to indirectly guide the GA to extract the true MCs and eliminate the false MCs (FMCs) accordingly. RESULTS AND CONCLUSIONS: The experimental results demonstrate that the incorporation of knowledge-discovery mechanism into the genetic algorithm is able to eliminate the FMCs and produce improved performance comparing with the conventional GA methods. Furthermore, the experimental results show that the proposed KD-GA method provides a promising and generic approach for the development of computer-aided diagnosis for breast cancer.

Algorithms↗

PubMed2005

Pretreatment anemia is correlated with the reduced effectiveness of radiation and concurrent chemotherapy in advanced head and neck cancer.

PURPOSE: Pretreatment anemia is an adverse prognostic variable in squamous cell head-and-neck cancer (HNC) patients treated with radiotherapy (RT) alone. Tumor hypoxia is an adverse parameter for treatment with RT alone or with RT and concurrent chemotherapy (CCT). Tumor hypoxia is more prevalent in patients who present with pretreatment hemoglobin (Hgb) concentrations less than 13 g/dL. RT/CCT improves survival over RT alone in advanced HNC, and its use is becoming more widespread. This study was performed to evaluate whether pretreatment Hgb less than 13 g/dL was correlated with treatment outcome in patients with advanced HNC treated with a uniform regimen of RT/CCT. METHODS AND MATERIALS: The study population consisted of patients with AJCC Stage III or IV, M0 HNC who were treated with 70 to 72.5 Gy accelerated hyperfractionated RT (1.25 Gy b.i.d.) and CCT consisting of 2 cycles of CDDP (12-20 mg/m(2)/d x 5 days) and continuous infusion 5-FU (600 mg/m(2)/d x 5 days) during Week 1 and Week 6. A planned break in RT occurred during Week 4. These patients were enrolled on the experimental arm of a prospective randomized trial that compared this regimen to hyperfractionated irradiation alone from 1990 to 1996. RT/CCT was delivered as standard therapy from 1996 to 2000. The primary endpoint was failure-free survival (FFS). Secondary endpoints included local-regional control and overall survival. RESULTS: One hundred and fifty-nine patients were treated from 1990 to 2000. The median (25-75%) pretreatment Hgb was 13.6 (12.2-13.5) g/dL. Hgb was 13 g/dL or higher in 105 patients and less than 13 g/dL in 54 patients. Primary tumor sites included oropharynx (43%), hypopharynx/larynx (36%), oral cavity (9%), and nasopharynx (6%). Seventy-eight percent of the patients with Hgb 13 g/dL or higher and 92% of the patients with Hgb less than 13 g/dL had a primary tumor stage of T3 or T4 (p = 0.01). Node-positive disease was present in 74 of 105 (70%) of patients with Hgb 13 g/dL or higher patients and in 36/54 (67%) of patients with Hgb less than 13 g/dL patients. Median follow-up of surviving patients was 42 months (range, 4-128 months). Five-year FFS was 75% for patients with Hgb 13 g/dL or higher vs. 50% for patients with Hgb less than 13 g/dL had a (p < 0.01). A total of 49 failures occurred in both patient cohorts. The median (25-75%) decrease in Hgb during RT/CCT was 2.2 (1.3-3.1) g/dL, both in patients who failed and in those who remained disease-free. CONCLUSION: Pretreatment Hgb less than 13 g/dL is correlated with adverse outcomes in advanced HNC patients treated with RT/CCT. Whether anemia actually causes poor outcomes remains unknown. The therapeutic effect of anemia correction is being evaluated in prospective trials.

Adult↗

PubMed2005

[Influencing factors for the curative effects of short-term continuous subcutaneous insulin infusion on newly diagnosed type 2 diabetes].

OBJECTIVE: To investigate the effects of short-term continuous subcutaneous insulin infusion (CSII) on newly diagnosed type 2 diabetes and to identify the influencing factors for the curative effects of CSII. METHODS: 138 newly diagnosed type 2 diabetic patients with fasting plasma glucose > 11.1 mmol/L were treated with CSII for 2 weeks. Intravenous glucose tolerance test (IVGTT) was performed before and after CSII. The target of glycemic control were fasting blood glucose < 6.1 mmol/L and postprandial blood glucose (PBG) < 8.0 mmol/L. The age, body mass index (BMI), fasting and postprandial plasma glucose, hemoglobin A(1C) (GHbA(1C)), Homa beta, Homa IR, area under the curve of insulin (AUC) during IVGTT were compared between the good glycemic control group and the inadequate glycemic control group. RESULTS: After 2 weeks' CSII treatment, good glycemic control was achieved in 126 patients (group A) but not in the remaining 12 patients (group B). There were no differences in age, BMI, postprandial plasma glucose, GHbA(1C), and Homa IR between the two groups before and after CSII treatment. But the fasting plasma glucose was higher and Homa B was lower in group B than in group A before CSII treatment. The DeltaAUC (AUC after CSII subtracted from that before CSII) representing the recovery of beta-cell function was much greater in group A than in group B. The insulin dose of group B was significantly higher than that of the good glycemic control group. CONCLUSION: More severe hyperglycemia and relative beta-cell function deficiency may be the main reasons responsible for not achieving good glycemic control in newly diagnosed type 2 diabetic patients with short-term intensive CSII treatment.

Adult↗

PubMed2005

A common pattern of persistent gene activation in human neocortical epileptic foci.

Epilepsy is a disease of recurrent seizures that can develop after a wide range of brain insults. Although surgical resection of focal regions of seizure onset can result in clinical improvement, the molecular mechanisms that produce and maintain focal hyperexcitability are not understood. Here, we demonstrate a regional, persistent induction of a common group of genes in human epileptic neocortex in 17 patients with neocortical epilepsy, regardless of the underlying pathology. This relatively small group of common genes, identified using complementary DNA microarrays and confirmed with quantitative reverse transcription polymerase chain reaction and immunostaining, include the immediate early gene transcription factors EGR-1, EGR-2, and c-fos, with roles in learning and memory, and signaling genes such as the dual-specificity kinase/phosphatase MKP-3. Maximal expression of these genes was observed in neurons in neocortical layers II through IV. These neurons also showed persistent cyclic adenosine monophosphate response element binding protein (CREB) activation and nuclear translocation of EGR-2 and c-fos proteins. In two patients, local interictal epileptiform discharge frequencies correlated precisely with the expression of these genes, suggesting that these genes either are directly modulated by the degree of epileptic activity or help sustain ongoing epileptic activity. The identification of a common set of genes and the persistent activation of CREB signaling in human epileptic foci provide a clinically relevant set of biological markers with potential importance for developing future diagnostic and therapeutic options in human epilepsy.

Adolescent↗

PubMed2005

Enhanced antitumor effect of the combination of tumstatin gene therapy and gemcitabine in murine models.

Targeting tumor endothelium is an important strategy for cancer therapy. We evaluated the effectiveness of gene therapy, that is, intramuscular delivery of plasmid DNA encoding tumstatin (pSecTag2B-tum), combined with gemcitabine administration in vitro and in vivo, using colon carcinoma (CT26) and Lewis lung carcinoma (LLC) murine models. The in vitro growth-inhibitory and proapoptotic effects of gemcitabine and/or tumstatin on human umbilical vein endothelial cells (HUVECs) and mouse endothelial cells (SVEC4-10), respectively, were assessed. in vitro, conditioned medium from pSecTag2B-tum-transfected COS cells inhibited the growth of endothelial cells but not of CT26 or LLC cells, whereas gemcitabine inhibited the growth of both endothelial cells and CT26 and LLC cells. Mice bearing subcutaneously established CT26 or LLC tumors received pSecTag2B-tum alone or in combination with gemcitabine to assess tumor growth inhibition. in vivo, combined treatment with pSecTag2B-tum and gemcitabine significantly decreased tumor growth through increased inhibition of tumor angiogenesis and increased tumor cell apoptosis compared with either agent alone. Enhanced antiproliferative and proapoptotic activity of the combination therapy on tumor-associated endothelial cells was calculated to be significant. This study suggests that combined treatment by the intramuscular delivery of plasmid DNA encoding tumstatin and gemcitabine augments tumor growth inhibition by suppressing angiogenesis and enhancing apoptosis in murine models. A combination of these agents could be used in future studies and translated into the clinical setting.

Angiogenesis Inhibitors↗

PubMed2005

[Change in plasma free 15 - F2t - isoprostane concentrations in smoking patients undergoing coronary artery bypass grafting and its clinical significance].

OBJECTIVE: To investigate the change in plasma free 15-F2t-isoprostane fn(15-F(2t)-IsoP) concentration and its relationship with postoperative cardiac function of smoking patients who underwent coronary artery bypass grafting (CABG). METHODS: Sixty patients with coronary artery heart disease were divided into smoking group [n=30, age 54-69 years old, American Heart Association (AHA)II-III] and non-smoking group(n=30, age 58-70 years old, ASA II-III). All the patients were scheduled for CABG under combination anesthesia with isoflurane and fentanyl and conventional cardiopulmonary bypass. Blood samples were drawn from the central vein to measure 15-F(2t)-IsoP at the following time points: before operation (T0); 30 minutes after aortic clamping for cardiopulmonary bypass(T1); 10 and 30 minutes after aortic declamping(T2,T3). RESULTS: There was no statistical difference in 15-F(2t)-IsoP between two groups at T0, but contents of 15-F(2t)-IsoP in the two groups was increased markedly at T1, compared to those at T0 (P<0.05 and P<0.01 for smoking and non-smoking groups, respectively), and the degree of increase of 15-F(2t)-IsoP in smoking group was significantly higher than that in non-smoking group(P<0.05). During reperfusion period (at T2,T3), the rate of lowering of 15-F(2t)-IsoP in smoking group was slower than that in non-smoking group(P<0.05), and moreover, these patients needed more inotropic support than those in non-smoking group. CONCLUSION: Smoking patients have weaker anti-oxidation capability during CABG, therefore they have higher incidence of low cardiac function.

Aged↗

PubMed2005

[Changes in content of inflammatory cytokines and troponin I in peri-operative periods of coronary artery bypass grafting with or without cardiopulmonary bypass].

OBJECTIVE: To evaluate the effects of coronary artery bypass grafting (CABG) with or without cardiopulmonary bypass (CPB) on inflammatory response and myocardial injury. METHODS: Sixty patients undergoing elective CABG were randomly divided into CABG with CPB group (CCABG, 30 cases) and CABG without CPB group (OPCABG, 30 cases). Blood samples were collected from central vein to assay interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), MB isoenzyme of creatine kinase (CK-MB) and troponin I (cTnI) before operation and 2, 8, 24, as well as 48 hours after operation, and all clinical data of the patients were recorded accordingly. RESULTS: After operation, the contents of IL-6, IL-8, TNF-alpha, CK-MB and cTnI were significantly higher in CCABG group than those before operation and those in OPCABG group, while most of these parameters showed little change in OPCABG group both in operative or postoperative period. CONCLUSION: The results suggest that OPCABG may provide better myocardial protection and less systemic inflammatory response than CCABG.

Aged↗

PubMed2005

[Recent advances in structures and relative enzyme properties of xylanase].

Xylanase can hydrolyze xylans into xylooligosaccharides and D-xylose, and has great prospect for applications in feed industry, paper and pulp industry, food industry and environment science. The study of xylanase had been started in 1960's. With the development and application of the new technologies, such as molecular biology, structural biology and protein engineering, many progresses have been made in the research of structures and functions of xylanase. This paper reviews the research progress and trend in the structure correlating with the important properties of xylanase. Analyses of three-dimensional structures and properties of mutants have revealed that glutamine and aspartic acid residues are involved in the catalytic mechanism. The thermostability of xylanase correlated with many factors, such as disulfide bridges, salt bridges, aromatic interactions, cotent of arginine and proline, and some multidomain xylanase have thermostability domains in N or C terminal. But no single mechanism is responsible for the remarkable stability of xylanase. The isoelectic points and reaction pH of xylanase are influenced by hydrophobicity and content of electric charges. Many researches had demonstrated that aromatic amino acid, histidine, and tryptophan play an important role in improving enzyme-substrate affinity. The researches of structures and functions of xylanase are of great significance in understanding the catalytic mechanism and directing the improvement of xylanase properties to meet the application requirement.

Catalysis↗

PubMed2005

[Expression of acidophilic alpha-amylase from Alicyclobacillus acidocaldarius].

The alpha-amylase (EC 3.2.1.1) from the Gram-positive Alicyclobacillus acidocaldarius was one kind of thermoacidophilic enzyme, with optimal temperature and pH of 75 degrees C and 3, respectively. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3901bp long, comprising one open reading frame encoding a polypeptide of 1301 amino acids. The calculated molecular weight of the alpha-amylase AMY was about 140kD. The gene amy was expressed in E. coli BL21 (DE3) and Pichia pastoris respectively, and both of the cloned proteins had bioactivity. The activity of amylase expressed in P. pastoris was further testified by amylase activity staining. The alpha-amylase expressed in P. pastoris had been purified and characterized. The apparent molecular weight of that was about 160kD according to SDS-PAGE. The optimum of pH for the enzyme was pH 3.2 as the native enzyme was; but the optimum of temperature was 65 degrees C and a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes in 70 degrees C. So the enzyme expressed by P. pastoris was also thermoacidophilic. Moreover some sequence was cloned by PCR, which ranged from + 1174 bp to + 3288 bp in the gene amy, encoding 705 amino acids with the calculated molecular weight of 79kD. The truncated gene amy' was expressed in E. coli BL21 (DE3) induced by 1 mmol/L IPTG, and the expressed enzyme also retained alpha-amylase activity.

Bacillus↗

PubMed2005

[Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties].

The lactase gene lacb' from Aspergillus candidus was fused behind alpha-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosylated protein with an apparent molecular weight of 130 kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110 kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6 mg/mL with enzymatic activity of 3600 U/mL in the 5 L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60 degrees C. The Vmax, Km, and specific activity of the lactase are 3.3 micromol/min, 1.7 mmol/L and 706.5 +/- 2.6 U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.

Aspergillus↗