Immobilization of α-amylase from anoxybacillus sp. SK3-4 on relizyme and immobead supports

α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0-9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 ?C. Immobilized TASKA retained 50% of its initial activity after 5-12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports

Carpooling with ecologists, geographers and taxonomists: perceptions from conducting environmental research in tropical regions

Greater than 80% of species on Earth are awaiting formal description, and simultaneously, many of these species unknown to science are becoming extinct. Here we highlight the importance and benefits of collaborating and working in interdisciplinary research groups, to improve quality and efficiency of both ecological and taxonomic research. The aim of this paper is to share and critique two methods used when conducting environmental field research in taxonomically data-poor parts of the world, such as Borneo. Through discussions with geographers, ecologists and taxonomists these two methods are evaluated. We conclude with a suggested solution to push taxonomic knowledge barriers by creating inter-disciplinary communities of researchers who work together to improve taxonomic identifications

Trapping of Intermediates with Substrate Analog HBOCaA in the Polymerizations Catalyzer by Class III Polyhydroxybutyrate (PHB) Synthase from Allochromatium Vinosum

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2–6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 hr−1. This extremely slow rate is due to thermodynamically unfavorable steps that involve formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2–3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [3H]-sT-PhaECAv and HBOCoA yielded [3H]-sTet-O-CoA at a rate constant faster than 17.4 s−1, which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s−1). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model

Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis

Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0–6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26–72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362–370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme

Effects of single and co-immobilization on the product specificity of type I pullulanase from Anoxybacillus sp. SK3-4

Type I pullulanase from Anoxybacillus sp. SK3-4 (PulASK) is an unusual debranching enzyme that specifically hydrolyzes starch α-1,6 linkages at long branches producing oligosaccharides (≥G8), but is nonreactive against short branches; thus, incapable of producing reducing sugars (G1–G7). We report on the effects of both single and co-immobilization of PulASK on product specificity. PulASK was purified and immobilized through covalent attachment to three epoxides (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Following immobilization, all PulASK derivatives were active on both short and long branches in starch producing reducing sugars (predominantly maltotriose) and oligosaccharides (≥G8), respectively, a feature that is absent in the free enzyme. This study also demonstrated that co-immobilization of PulASK and α-amylase from Anoxybacillus sp. SK3-4 (TASKA) on ReliZyme HFA403/M significantly changed the product specificity compared to the free enzymes alone or individually immobilized enzymes. In conclusion, individual or co-immobilization caused changes in the product specificity, presumably due to changes in the enzyme binding pocket caused by the influence of carrier surface properties (hydrophobic or hydrophilic) and the lengths of the spacer arms

Diagnostic Value of 14C Urea Breath Test for Helicobacter pylori Detection Compared by Histopathology in Indonesian Dyspeptic Patients

Muhammad Miftahussurur,1,2 Adinta Windia,1 Ari Fahrial Syam,3 Iswan Abbas Nusi,1 Ricky Indra Alfaray,2,4 Kartika Afrida Fauzia,2,4 Hartono Kahar,5 Herry Purbayu,1 Titong Sugihartono,1 Poernomo Boedi Setiawan,1 Ummi Maimunah,1 Ulfa Kholili,1 Husin Thamrin,1 Amie Vidyani,1 Dalla Doohan,2 Langgeng Agung Waskito,2 Yudith Annisa Ayu Rezkitha,2,6 Gontar Alamsyah Siregar,7 Yoshio Yamaoka1,4 1Gastroentero-Hepatology Division, Department of Internal Medicine, Faculty of Medicine, Universitas Airlangga, Surabaya, East Java, 60286, Indonesia; 2Institute of Tropical Disease, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia; 3Division of Gastroenterology, Department of Internal Medicine, Faculty of Medicine, University of Indonesia, Jakarta, 10430, Indonesia; 4Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu, 879-5593, Japan; 5Department of Clinical Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, East Java, 60286, Indonesia; 6Faculty of Medicine, University of Muhammadiyah Surabaya, Surabaya, East Java, 60113, Indonesia; 7Division of Gastroentero-Hepatology, Department of Internal Medicine, Faculty of Medicine, University of Sumatra Utara, Medan, 20155, IndonesiaCorrespondence: Muhammad MiftahussururGastroentero-Hepatology Division, Department of Internal Medicine, Faculty of Medicine, Dr. Soetomo Teaching Hospital, Universitas Airlangga, Jalan Mayjend Prof. Dr. Moestopo No. 6– 8, Surabaya, 60286, IndonesiaTel/Fax +6231-502-3865Email [email protected]: Histopathology method is often used as a gold standard diagnostic for Helicobacter pylori infection in Indonesia. However, it requires an endoscopic procedure which is limited in Indonesia. A non-invasive method, such as 14C Urea Breath Test (UBT), is more favorable; however, this particular method has not been validated yet.Patients and Methods: A total of 55 dyspeptic patients underwent gastroscopy and 14C-UBT test. We used Heliprobe® UBT for UBT test. As for the histology, May-Giemsa staining of two gastric biopsies (from the antrum and corpus) were evaluated following the Updated Sydney System.Results: The Receiver Operating Characteristics analysis showed that the optimum cut-off value was 57 with excellence Area under Curve = 0.955 (95% CI = 0.861– 1.000). By applying the optimum cut-off value, Heliprobe® UBT showed 92.31% for sensitivity, 97.62% for specificity, 92.31% for positive predictive value, 97.62% for negative predictive value, 38.77 for positive likelihood ratio, 0.0788 for negative likelihood ratio, and 96.36% for the accuracy.Conclusion: The 14C-UBT is an accurate test for H. pylori diagnosis with excellent sensitivity, specificity, and accuracy. The different optimum cut-off points suggested that a validation is absolutely necessary for new test prior application to the new population.Keywords: Helicobacter pylori, UBT, diagnostic, infectious diseas