Cloning and Nucleotide Analysis of a Mannanase Gene from Bacillus sp. GA2(1)

บทคัดย่อ งานวิจัยนี้ศึกษายีนแมนนาเนสจาก Bacillus sp. GA2(1) ที่คัดแยกได้จากบริเวณที่มีการทิ้งกากถั่วเหลืองในจังหวัดขอนแก่น ประเทศไทย โดยออกแบบคู่ไพร์เมอร์ที่จำเพาะต่อปลายด้าน 5’ และ 3’ ของยีนแมนนาเนสจากแบคทีเรียสายพันธุ์ใกล้เคียงที่มีการรายงานลำดับนิวคลีโอไทด์ในฐานข้อมูล GenBank เพื่อนำมาเพิ่มปริมาณยีนด้วยปฏิกิริยาลูกโซ่พอลิเมอเรสและใช้จีโนมิกดีเอ็นเอเป็นดีเอ็นเอแม่แบบ จากการศึกษาพบว่ายีนแมนนาเนสมีขนาดประมาณ 1,100 คู่เบส เมื่อแปลลำดับนิวคลีโอไทด์ที่ได้เป็นลำดับกรดอะมิโน และวิเคราะห์ผลด้วยโปรแกรม ProtParam พบว่าแมนนาเนสจาก Bacillus sp. GA2(1) ชนิดนี้มีมวลโมเลกุลเท่ากับ 42.08 กิโลดาลตัน และมีค่า pI เท่ากับ 6.04 และจากการวิเคราะห์โครงสร้างของเอนไซม์ด้วยโปรแกรม PROSITE และ InterPro พบว่ารีคอมบิแนนท์แมนนาเนสนี้จัดอยู่ในกลุ่ม glycosyl hydrolases สกุล 26 (GH26) ซึ่งมีสมบัติเป็น mannan endo-1,4-beta-mannosidase ที่สามารถไฮโดรไลซ์ (1->4)-beta-D-mannosidic linkagesในแมนแนนและกาแลคโตแมนแนนได้คำสำคัญ: Bacillus sp. ยีนแมนนาเนส Glycosyl hydrolases 26 ABSTRACTWe studied a mannanase gene from Bacillus sp. GA2(1) isolated from soybean meal in Khon Kaen province, Thailand. A pair of primers was designed from 5’ and 3’ end of mannanase genes of closely related species in which the nucleotide sequences were available in the GenBank database. The polymerase chain reaction was then carried out using genomic DNA as a template. The PCR product with the size of approximately 1,100 base pairs was obtained and submitted for sequencing. Using ProtParam program, the predicted Mw of the mannanase from Bacillus sp. GA2(1) was 42.08 kDa with the pI value of 6.04. Structural prediction by PROSITE and InterPro program suggested that this mannanase belonged to glycosyl hydrolases family 26 (GH26). The members of this enzyme family possessed mannan endo-1,4-beta-mannosidase activity in that they were able to hydrolyze (1®4)-beta-D-mannosidic linkages in mannans and galactomannans.Keyword: Bacillus sp., mannanase gene, Glycosyl hydrolases 26

Optimization of extracellular mannanase production from Penicillium oxalicum KUB-SN2-1 and application for hydrolysis property

Effects of media composition, and physical properties on the production of crude mannanase by Penicillium oxalicumKUB-SN2-1 were investigated. P.oxalicum KUB-SN2-1 was propagated in a shaking incubator at 30°C with rotation speed of200 rpm of 7 days. The specific activity obtained during growth on robusta coffee residues (RCR) of 16.21 U/mg protein wasmuch higher than other carbon sources tested. For nitrogen sources, yeast extract (0.11 U/mg protein) and ammonium nitrate(0.09 U/mg protein) showed maximum specific activity. Hence, guar gum was the best inducer for producing mannanase (14U/mg protein). For evaluating the optimal concentration, the result showed that 1% guar gum, 0.5% yeast extract, 0.25%ammonium nitrate, and 0.25% RCR were the suitable sources of inducer, organic nitrogen, inorganic nitrogen, and carbon,respectively. Modified medium with initial culture pH of 5.0 at 30°C was optimum for mannanase production (53.77 U/ml for3 day). Reducing sugars were analyzed by dinitrosalicylic acid methods. The highest reducing sugar of 7517.82 g/mlwas obtained from copra meal hydrolysate after 30 h

Why Do Livestock Farmers Participate in Voluntary Environmental Programs? An Empirical Study of the Michigan Agriculture Environmental Assurance Program (MAEAP)

Previous studies have identified regulatory preemption and differentiation as two main motives for participation in voluntary environmental programs (VEPs). This research examines the motivations of livestock farmers to participate in the Michigan Agricultural Environmental Assurance Program (MAEAP). It employs a signaling model of interaction between the regulator and livestock farmers under imperfect information to analyze the potential equilibria of participation decisions in VEPs. Data from a survey of livestock farmers in Michigan is analyzed to test hypotheses regarding the realized equilibrium in MAEAP participation. The results suggest that livestock farmers who are interested in regulatory preemption are more likely to be MAEAP-verified while those who are interested in differentiation are less likely to be verified at the time of the survey. Consistent with the model predictions under the regulatory preemption equilibrium, MAEAP-certified farms also perceived more stringent enforcement effort by the regulators

Enhancing Motivation in Online Courses with Mobile Communication Tool Support: A Comparative Study

Mobile technologies have helped establish new channels of communication among learners and instructors, potentially providing greater access to course information, and promoting easier access to course activities and learner motivation in online learning environments. The paper compares motivation between groups of learners being taught through an online course based on an e-learning system with and without the support of mobile communication tools, respectively. These tools, which are implemented on a mobile phone, extend the use of the existing Moodle learning management system (LMS) under the guidance of a mobile communication tools framework. This framework is considered to be effective in promoting learner motivation and encouraging interaction between learners and instructors as well as among learner peers in online learning environments. A quasi-experimental research design was used to empirically investigate the influence of these tools on learner motivation using subjective assessment (for attention, relevance, confidence, satisfaction, and social ability) and objective assessment (for disengagement, engagement, and academic performance). The results indicate that the use of the tools was effective in improving learner motivation, especially in terms of the attention and engagement variables. Overall, there were statistically significant differences in subjective motivation, with a higher level achieved by experimental-group learners (supported by the tools) than control-group learners (unsupported by the tools)

Evaluating the Prebiotic Properties of Agar Oligosaccharides Obtained from the Red Alga <i>Gracilaria fisheri</i> via Enzymatic Hydrolysis

Currently, the demand in the food market for oligosaccharides with biological activities is rapidly increasing. In this study, agar polysaccharides from Gracilaria fisheri were treated with β-agarases and hydrolyzed to agar oligosaccharides (AOSs). High-performance anion-exchange chromatography/pulsed amperometric detection (HPAEC-PAD), Fourier-transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC), were employed to analyze the chemical characteristics of AOSs. The FT-IR spectra revealed that the enzymatic hydrolysis had no effect on specific functional groups in the AOS molecule. To investigate the prebiotic and pathogen inhibitory effects of AOSs, the influence of AOSs on the growth of three probiotic and two pathogenic bacteria was examined. The gastrointestinal tolerance of probiotics in the presence of AOSs was also investigated. AOSs enhanced the growth of Lactobacillus plantarum by 254%, and inhibited the growth of Bacillus cereus by 32.80%, and Escherichia coli by 58.94%. The highest survival rates of L. plantarum and L. acidophilus were maintained by AOSs in the presence of α-amylase and HCl under simulated gastrointestinal conditions. This study demonstrates that AOSs from G. fisheri exhibit potential as a prebiotic additive in foods

Poikiloderma with neutropenia and associated squamous cell carcinoma: A case report

Here, we describe a case of a patient with known poikiloderma with neutropenia who developed cutaneous squamous cell carcinoma in a chronically sun-exposed area at the age of 14. To date, there is only one other report of this association. This report highlights the need for routine skin cancer screening in patients with this diagnosis as well as the importance of a correct initial diagnosis

Characterization of mannanase S1 from Klebsiella oxytoca KUB-CW2-3 and its application in copra mannan hydrolysis

ABSTRACT: The mannanase S1 from Klebsiella oxytoca KUB-CW2-3 was purified by a single anion exchange chromatography to electrophoretic homogeneity. S1 is a protein with a molecular mass of approximately 165 kDa and a pI value of 3.5. The optimum pH and temperature of mannanase activity were 4.0 and 40°C, respectively. The enzyme exhibited good stability over the broad pH range of 3-6. The mannanase S1 exhibited specific activity for different mannan substrates including galactomannan from locust bean gum (LBG), copra meal, and glucomannan from konjac, while neither xylanase nor cellulase activity were detectable. The Michaelis-Menten constants (Km), maximum velocity (Vmax), and the catalytic constant (kcat) values of S1 against LBG and konjac mannan were 1.038-1.056 mg/ml, 6.149-6.183 µU ml −1 min −1 , and 0.047 s −1 , respectively. In addition, mannanase activity was activated by Co 2+ (129%) but completely inhibited by EDTA and Zn 2+ . The N-terminal amino acid sequence (GRVGEAGPHGPHGPH) of mannanase S1 is different from the N-terminal region of other bacterial mannanases belonging to the glycoside hydrolase family GH5. The degradation products of mannanase S1 from LBG hydrolysis were galactose and unknown oligosaccharides with a different molecular structure to mannobiose, triose, and tetraose indicating the cleavages of α-1,6-galactosidic and β-1,4-mannosidic linkages. Its hydrolysis of 100 mM CoCl2-treated copra mannan enhanced the growth of Lactobacillus reuteri KUB-AC5 but inhibited that of Salmonella serovar Enteritidis S003, indicating potential prebiotic properties by the action of mannanase from K. oxytoca