Journal articles: 'Glulam frame'

1

Buchanan, A. H., and R. H. Fairweather. "Seismic design of glulam structures." Bulletin of the New Zealand Society for Earthquake Engineering 26, no. 4 (December 31, 1993): 415–36. http://dx.doi.org/10.5459/bnzsee.26.4.415-436.

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This paper gives an overview of the seismic performance of glue laminated (glulam) timber frame buildings. It describes the wide range of connections that can be used in glulam frames, for both single storey and multi-storey buildings, with particular reference to seismic loading. Several new connections incorporating epoxied steel bars are described in detail. Testing of these connections under simulated seismic loading is reported, with recommendations for seismic design. A design procedure is given for low rise multi-storey glulam frame buildings.

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Sakata, Hiroyasu, Hiroshi Abe, and Hiromichi Ito. "Moment Resisting Finger Joints of Glulam Frame Knees." IABSE Symposium Report 85, no. 1 (January 1, 2001): 55–60. http://dx.doi.org/10.2749/222137801796349231.

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Zonta, Daniele, Cristiano Loss, Maurizio Piazza, and Paolo Zanon. "Direct Displacement-Based Design of Glulam Timber Frame Buildings." Journal of Earthquake Engineering 15, no. 3 (March 31, 2011): 491–510. http://dx.doi.org/10.1080/13632469.2010.495184.

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Guo, Nan, Chao Yang, Ling Li, Guodong Li, and Yan Zhao. "Experimental Study on Flexural Performance of Regulated Reinforced Glulam Beam after Long-Term Loading." Sustainability 13, no. 10 (May 17, 2021): 5556. http://dx.doi.org/10.3390/su13105556.

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Due to wood creep characteristics, the failure mode, bearing capacity, stiffness, and deformation of its components are doomed to be impacted by long-term loading. This paper conducted a comparative test on creep beams, regulated beams, and short-term beams based on the former long-term loading research. The results demonstrated that the glulam beam experienced tensile failure of the beam-bottom, while the horizontal joint failure and the local compressive failure of the beam-end happened in the reinforced glulam beam and the prestressed glulam beam. The bearing capacity of the creep beams decreased compared with that of the short-term beams; the decline in the bearing capacity of the ordinary glulam beams, the reinforced glulam beams, and the prestressed glulam beams ranged from 3.2% to 9.8%, from 1.6% to 13.2%, and from 2.9% to 9.2%, respectively. However, the bearing capacity of the regulated beam with the deformation restored to the initial value of the load increased by 4.6–14.1%. The prestressed regulation changed the distribution of the stress on the beam and thus enhanced its bearing capacity. The findings of this work could be used as a frame of reference for similar components in engineering applications.

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Komatsu, Kohei, and Akihisa Kitamori. "Static and Dynamic Properties of Portal Frames Composed of Built-Up Sawn Square Timber." Wood Research Journal 3, no. 1 (August 27, 2017): 36–43. http://dx.doi.org/10.51850/wrj.2012.3.1.36-43.

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In order to propose an alternative structural element to be used for wooden dwelling houses in rich forest area, we paid attentions to a portal frame structure which is composed of not glulam but built-up members whose raw materials are dried sawn timbers taken from plantation grown forest. For establishing design procedure of the structural element, we made two different types of portal frames and conducted, at first, basic dynamic test to estimate natural frequency and dumping factors by fixing small shake excitation machine on the portal frames, then static push-pull cyclic loading tests were conducted until failure. The natural frequency of both portal frames was almost same but the higher order frequencies were likely to be affected by the difference of shear reinforcement of built-up members by hardwood dowels. While on static properties, as both portal frames failed in brittle manner due to bending failure at column or tear off at connection plate made of compression wood, further improvement of connection system was required for obtaining more stable alternative elements to glulam.

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Zheng, Wei, Weidong Lu, Weiqing Liu, and Yue Li. "Lateral loading behavior of glulam frame-midply hybrid lateral systems." Construction and Building Materials 220 (September 2019): 53–63. http://dx.doi.org/10.1016/j.conbuildmat.2019.05.182.

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Ding, Yi, Zhen Zhou, Linjie Huang, and Yi Si. "Seismic performance of self-centering glulam frame with friction damper." Engineering Structures 245 (October 2021): 112857. http://dx.doi.org/10.1016/j.engstruct.2021.112857.

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8

Zhao, Xuan, Binsheng Zhang, Tony Kilpatrick, Iain Sanderson, and Dewen Liu. "Numerical Analysis on Global Serviceability Behaviours of Tall Glulam Frame Buildings to the Eurocodes and UK National Annexes." Journal of Civil Engineering and Construction 10, no. 3 (August 15, 2021): 109–22. http://dx.doi.org/10.32732/jcec.2021.10.3.109.

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Glued-laminated timber (Glulam) is an innovative engineered timber product and has been widely used for constructing spatial grand timber structures and tall timber buildings due to its exceptional natural attraction, easy processing, decent fire resistance and outstanding structural performance. However, global serviceability performances of tall timber buildings constructed from Glulam products for beams, columns and bracings and CLT products for lift core and floors under wind load are not well known yet though they are crucial in structural design and global analysis. In this study, finite element software SAP2000 is used to numerically simulate the global static and dynamic serviceability behaviours of a 105 m high 30-storey tall Glulam building with CLT lift core and floors assumed in Glasgow, Scotland, UK. The maximum horizontal storey displacement due to wind is 58.5% of the design limit and the maximum global horizontal displacement is 49.7% of the limit set to the Eurocodes. The first three lowest vibrational frequencies, modes and shapes of the building are obtained, with the fundamental frequency being 33.3% smaller than the code recommended value due to its low mass and stiffness. The peak acceleration of the building due to wind is determined to the Eurocodes and ISO 10137. The results show that the global serviceability behaviours of the building satisfy the requirements of the Eurocodes and other design standards. Parametric studies on the peak accelerations of the tall Glulam building are also conducted by varying timber material properties and building masses. Increasing the timber grade for CLT members, the generalised building mass and the generalised building stiffness can all be adopted to lower the peak accelerations at the top level of the building so as to reduce the human perceptions to the wind induced vibrations with respect to the peak acceleration.

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Holzer, S. M., C. H. Wu, and J. Tissaoui. "Finite Element Stability Analysis of a Glulam Dome." International Journal of Space Structures 7, no. 4 (December 1992): 353–61. http://dx.doi.org/10.1177/026635119200700411.

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The paper centres on stability investigations of a glued-laminated timber (glulam) dome under several snow load conditions. The dome consists of a triangulated network of curved glulam beams, a decking supported by curved purlins, and a steel tension ring. The dome is represented by two different models. The first model is a rigid-jointed space frame composed of curved beam elements. The second model consists of straight beam elements, with rigid or flexible joints, and a bracing to simulate the lateral support of the beams provided by the decking. Two finite element methods are presented and used in the analyses: A nonlinear method that computes the buckling load and a combined nonlinear/linear eigenvalue method that provides estimates of the buckling load. The results presented include buckling pressures, buckling modes, effects of joint stiffness and bracing on the stability of the dome, and the status of the material prior to buckling.

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MIYAZAWA, Kenji. "STUDY ON STRESS BEHAVIOR AND DUCTILITY OF GLULAM FRAME STRUCTURES WITH PLYWOOD SHEAR WALLS." Journal of Structural and Construction Engineering (Transactions of AIJ) 65, no. 527 (2000): 125–32. http://dx.doi.org/10.3130/aijs.65.125_1.

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Wu, Guofang, Yong Zhong, Yingchun Gong, and Haiqing Ren. "Application of Modern Wood Product Glulam in Timber Frame With Tenon-Mortise Joints and Its Structural Behavior." Journal of Renewable Materials 7, no. 5 (2019): 451–61. http://dx.doi.org/10.32604/jrm.2019.06229.

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Petrycki, Adam, and Osama “Sam” Salem. "Structural Integrity of Bolted Glulam Frame Connections Reinforced with Self-Tapping Screws in a Column Removal Scenario." Journal of Structural Engineering 146, no. 10 (October 2020): 04020213. http://dx.doi.org/10.1061/(asce)st.1943-541x.0002792.

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van de Kuilen, Jan Willem G., and Wolfgang Gard. "Damage Assessment and Residual Service Life Estimation of Cracked Timber Beams." Advanced Materials Research 778 (September 2013): 402–9. http://dx.doi.org/10.4028/www.scientific.net/amr.778.402.

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Service life modelling of cracked timber beams can be performed using modified damage accumulation models that describe the combined effect or long term loads (mechanical) and biological of physical wood degradation. The combined model allows for the estimation of residual service life and an analysis of crack development. The model can also be used to analyse safety factors that may need to be applied. Also, a sensitivity analysis can be performed for future risks. It is shown that the failure risk is very sensitive to the level of the applied loads, similar to time to failure analysis of non-degraded timber. Failure in timber structures occurs within a very short time frame. A practical case of cracked glulam beams is included in the paper.

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TAKAHASHI, Shigeo, Toshio KITAMURA, Takuro MORI, Akira SASAGAWA, and Hiroshi ISODA. "RELATIONSHIP AND SEASONAL INFLUENCE OF THE CREEP BEHAVIOR ON GLULAM MEMBER, JOINT AND FRAME UNDER NON-CONTROLLED CONDITION." Journal of Structural and Construction Engineering (Transactions of AIJ) 67, no. 551 (2002): 87–94. http://dx.doi.org/10.3130/aijs.67.87_1.

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Boscato, Giosuè, Alessandra Dal Cin, and Riccardo Destro. "Structural Behaviour and Comparison of CGF Panels." Advanced Materials Research 900 (February 2014): 463–67. http://dx.doi.org/10.4028/www.scientific.net/amr.900.463.

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A CGF Panel (Concrete Glulam Framed Panel) is a concrete panel with a glued laminated timber frame. The experimentation on this new construction system at LabSCo (Laboratory of Strength of Materials) of IUAV University of Venice, inspired a wide research on buildings made of this construction system investigating in different aspect of building behaviour: particularly about mechanical property of the materials, mechanical of the system and building physics. This paper presents the results of quasi-static in-plane tests on single panel and configurations of some different panels. The tests in the laboratory are used for measuring the in-plane strength and stiffness of individual panels and wall sections consist of some panels in order to verify and measure the behavior of the connections between the various parts of the single panel and the connection between the panels. Thanks to the results obtained it was possible carry out the FE model to calibrate the characteristics in relation to experimental data. Finally, in order to compare this constructive system with the well known X-lam systems, on the basis of the calibration of the models we were able to set up a comparable FE model with those of the X-lam wall described in the publication: "Quasi-Static and Pseudo-Dynamic Tests on XLAM Walls and Buildings " inherent in the SOFIE project coordinated by the CNR-IVALSA (Italian National Research Council - Trees and Timber Institute)

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Ormarsson, Sigurdur, and Óskar V. Gíslason. "Moisture-induced stresses in glulam frames." European Journal of Wood and Wood Products 74, no. 3 (March 1, 2016): 307–18. http://dx.doi.org/10.1007/s00107-016-1006-5.

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17

Royles, R., and E. J. Mullen. "Splice jointing behaviour in glulam portal frames." Strain 34, no. 1 (February 1998): 19–23. http://dx.doi.org/10.1111/j.1475-1305.1998.tb01071.x.

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Popovski, Marjan, Helmut G. L. Prion, and Erol Karacabeyli. "Seismic performance of connections in heavy timber construction." Canadian Journal of Civil Engineering 29, no. 3 (June 1, 2002): 389–99. http://dx.doi.org/10.1139/l02-020.

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Results from monotonic and quasi-static cyclic tests on connections used in heavy timber construction are presented for two types of fasteners: steel bolts and glulam rivets. Bolted connections with three different diameter bolts, arranged in several configurations, as well as two different configurations of glulam rivet connections were tested. All configurations included a main glulam member and two steel side plates. For bolted connections, the seismic behaviour was found to be primarily dependent on the bolt slenderness ratio. Bolted connections with higher slenderness ratios (smaller diameter bolts) exhibited more ductile behaviour with considerable steel yielding and wood crushing before failure. Glulam riveted connections, which were designed in rivet failure mode, showed superior seismic performance when compared to bolted connections for similar design load levels. Riveted connections were also able to dissipate the highest amount of input energy before the failure was reached.Key words: timber connections, glulam rivets, bolts, ductility, timber, wood, braced frames, seismic performance, heavy timber construction.

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Dong, Wenchen, Minghao Li, Chin-Long Lee, and Gregory MacRae. "Numerical modelling of glulam frames with buckling restrained braces." Engineering Structures 239 (July 2021): 112338. http://dx.doi.org/10.1016/j.engstruct.2021.112338.

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Zhong, Li Li, Chun Yu Wei, Yan Xiao, and Bo Shan. "The Regional Expression in Utilization of Material Construction: The Landscape Sketch Practice of the Modern Bamboo Structures in Meixi Lake Project." Key Engineering Materials 517 (June 2012): 150–57. http://dx.doi.org/10.4028/www.scientific.net/kem.517.150.

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Based on the practice of the landscape sketch bamboo buildings of Meixi Lake in Changsha, this thesis, from the perspective of material construction, aims to perceive the region and bamboo structure, to concern the bamboo frame form and space construction, to try to integrate bamboo structure with other materials and to explore the formation of bamboo-framed building and the related regional expression. Excavating bamboo value, improving bamboo performance and developing bamboo structural building can satisfy requirements as the main part of construction and expression in architecture and promote the using level of GluBam. Technological innovation such as suitable construction skills and reasonable structural system can promote the effective utilizing bamboo and development of modern bamboo structure. It is a alternative green building system for sustainable development.

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Dong, Wenchen, Minghao Li, Chin-Long Lee, Gregory MacRae, and Anthony Abu. "Experimental testing of full-scale glulam frames with buckling restrained braces." Engineering Structures 222 (November 2020): 111081. http://dx.doi.org/10.1016/j.engstruct.2020.111081.

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22

Roset, Mara S., Andrés E. Ciocchini, Rodolfo A. Ugalde, and Nora Iñón de Iannino. "The Brucella abortus Cyclic β-1,2-Glucan Virulence Factor Is Substituted with O-Ester-Linked Succinyl Residues." Journal of Bacteriology 188, no. 14 (July 15, 2006): 5003–13. http://dx.doi.org/10.1128/jb.00086-06.

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ABSTRACT Brucella periplasmic cyclic β-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic β-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic β-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic β-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-β-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection.

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Chen, Rongji, Arvind A. Bhagwat, Robert Yaklich, and Donald L. Keister. "Characterization ofndvD, the third gene involved in the synthesis of cyclic β-(13),(16)-D-glucans inBradyrhizobium japonicum." Canadian Journal of Microbiology 48, no. 11 (November 1, 2002): 1008–16. http://dx.doi.org/10.1139/w02-099.

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Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic β-(1[Formula: see text]3),(1[Formula: see text]6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for β-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo β-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral β-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for β-glucan synthesis. Our hypothesis for the mechanism of β-(1[Formula: see text]3),(1[Formula: see text]6)-D-glucan synthesis is presented. Key Words: β-glucans,Bradyrhizobium, soybean, nitrogen fixation.

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Roset, Mara S., Andrés E. Ciocchini, Rodolfo A. Ugalde, and Nora Iñón de Iannino. "Molecular Cloning and Characterization of cgt, the Brucella abortus Cyclic β-1,2-Glucan Transporter Gene, and Its Role in Virulence." Infection and Immunity 72, no. 4 (April 2004): 2263–71. http://dx.doi.org/10.1128/iai.72.4.2263-2271.2004.

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ABSTRACT The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic β-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic β-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic β-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic β-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.

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Михайловський, Денис Віталійович, Дмитро Миколайович Матющенко, and Артур Олегович Смоленський. "Influence of uneven settlements of the curved glulam frames’ bearings on the cornice node’s stress-strain state." ScienceRise 7, no. 2 (24) (July 30, 2016): 25. http://dx.doi.org/10.15587/2313-8416.2016.74484.

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Jung, Hong-Ju, Yo-Jin Song, In-Hwan Lee, and Soon-Il Hong. "Lateral Load Performance Evaluation of Larch Glulam Portal Frames Using GFRP-Reinforced Laminated Plate and GFRP Rod." Journal of the Korean Wood Science and Technology 44, no. 1 (January 25, 2016): 30–39. http://dx.doi.org/10.5658/wood.2016.44.1.30.

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Iñón de Iannino, Nora, Gabriel Briones, Marcelo Tolmasky, and Rodolfo A. Ugalde. "Molecular Cloning and Characterization of cgs, theBrucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB andAgrobacterium tumefaciens chvB Mutants." Journal of Bacteriology 180, no. 17 (September 1, 1998): 4392–400. http://dx.doi.org/10.1128/jb.180.17.4392-4400.1998.

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ABSTRACT The animal pathogen Brucella abortus contains a gene,cgs, that complemented a Rhizobium melilotinodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved inRhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor inBrucella infection.

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Deutsch, Stéphanie-Marie, Pierre Le Bivic, Christophe Hervé, Marie-Noëlle Madec, Gisèle LaPointe, Gwenaël Jan, Yves Le Loir, and Hélène Falentin. "Correlation of the Capsular Phenotype in Propionibacterium freudenreichii with the Level of Expression of gtf, a Unique Polysaccharide Synthase-Encoding Gene." Applied and Environmental Microbiology 76, no. 9 (March 12, 2010): 2740–46. http://dx.doi.org/10.1128/aem.02591-09.

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ABSTRACT Many food-grade bacteria produce exopolysaccharides (EPS) that affect the texture of fermented food products and that may be involved in probiotic properties. Propionibacterium freudenreichii is a Gram-positive food-grade bacterium with reported probiotic capabilities that is widely used as starter in Swiss-type cheese. In this study, 68 strains of P. freudenreichii were screened for the β-glucan capsular phenotype by immunoagglutination with a specific antibody and for the presence of the gtf gene coding for polysaccharide synthase. All strains were positive for PCR amplification with gtf gene-specific primers, but the presence of β-glucan capsular EPS was detected for only 35% of the strains studied. Disruption of gtf in P. freudenreichii revealed that gtf is a unique gene involved in β-glucan capsular EPS production in P. freudenreichii. The gtf gene was transferred into and expressed in Lactococcus lactis, in which it conferred an agglutination-positive phenotype. Expression of the gtf gene was measured by performing quantitative reverse transcription-PCR assays with RNA from four capsular and three noncapsular strains. A positive correlation was found between the β-glucan capsular phenotype and gtf gene expression. Sequencing of the region upstream of the gtf open reading frame revealed the presence of an insertion element (IS element) in this upstream region in the four strains with the β-glucan capsular phenotype. The role of the IS element in the expression of neighboring genes and its impact on interstrain variability of the P. freudenreichii capsule phenotype remain to be elucidated.

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Kralj, S., G. H. van Geel-Schutten, H. Rahaoui, R. J. Leer, E. J. Faber, M. J. E. C. van der Maarel, and L. Dijkhuizen. "Molecular Characterization of a Novel Glucosyltransferase from Lactobacillus reuteri Strain 121 Synthesizing a Unique, Highly Branched Glucan with α-(1→4) and α-(1→6) Glucosidic Bonds." Applied and Environmental Microbiology 68, no. 9 (September 2002): 4283–91. http://dx.doi.org/10.1128/aem.68.9.4283-4291.2002.

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ABSTRACT Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the α-(1→4) glucosidic type. The glucan also contains α-(1→6)-linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M r of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.

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Chia, Jean-San, Czau-Siung Yang, and Jen-Yang Chen. "Functional Analyses of a Conserved Region in Glucosyltransferases of Streptococcus mutans." Infection and Immunity 66, no. 10 (October 1, 1998): 4797–803. http://dx.doi.org/10.1128/iai.66.10.4797-4803.1998.

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ABSTRACT Streptococcus mutans glucosyltransferases (GTFs; GtfB, -C, and -D) synthesize water-soluble and -insoluble glucan polymers from sucrose. We have identified previously a conserved region of 19 amino acids (aa) (Gtf-P1; aa 409 to 427 of GtfB and aa 435 to 453 of GtfC) which is functionally important for both enzymatic activity and bacterial adherence. Monoclonal antibodies directed against Gtf-P1 selectively inhibited insoluble glucan synthesis by GtfB and -C but had no effect on soluble glucan synthesis by GtfD, suggesting that despite an apparent near identity of sequence, corresponding residues may function differently in these enzymes. To test this hypothesis, we used different strategies of mutagenesis to analyze amino acid residues of GtfB and GtfC in Gtf-P1. In-frame insertion of 6 amino acids preceding, or deletion of 14 amino acids within, this conserved region abolished the enzymatic activities of both GtfB and GtfC. Substitution of several residues in combination by random mutagenesis resulted in GtfB, but not GtfC, enzymes exhibiting decreased glucan synthesis and reduced rates of sucrose hydrolysis. Amino acid substitutions of Asp residues in GtfB or GtfC were found to be more critical for enzymatic activity than at other positions of this region. Interestingly, single mutation at Asp411 or Asp413 of GtfB resulted in enzymes retaining about 20% of wild-type activity, whereas mutagenesis of the corresponding Asp at position 437 or 439 in GtfC resulted in complete loss of enzymatic activity. Furthermore, single amino acid substitution of a Val residue between the two Asp residues enhanced the sucrase- and glucan-synthesizing activities of GtfB and GtfC. These results confirmed the report from another laboratory that Asp residues in the Gtf-P1 region are essential for enzymatic catalysis and provide new evidence that identical residues may function differently in closely related Gtf enzymes.

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Hong, Z., P. Mann, N. H. Brown, L. E. Tran, K. J. Shaw, R. S. Hare, and B. DiDomenico. "Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis." Molecular and Cellular Biology 14, no. 2 (February 1994): 1017–25. http://dx.doi.org/10.1128/mcb.14.2.1017.

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k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

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32

Hong, Z., P. Mann, N. H. Brown, L. E. Tran, K. J. Shaw, R. S. Hare, and B. DiDomenico. "Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis." Molecular and Cellular Biology 14, no. 2 (February 1994): 1017–25. http://dx.doi.org/10.1128/mcb.14.2.1017-1025.1994.

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k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

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33

Comfort, Donald A., Chung-Jung Chou, Shannon B. Conners, Amy L. VanFossen, and Robert M. Kelly. "Functional-Genomics-Based Identification and Characterization of Open Reading Frames Encoding α-Glucoside-Processing Enzymes in the Hyperthermophilic Archaeon Pyrococcus furiosus." Applied and Environmental Microbiology 74, no. 4 (December 21, 2007): 1281–83. http://dx.doi.org/10.1128/aem.01920-07.

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ABSTRACT Bioinformatics analysis and transcriptional response information for Pyrococcus furiosus grown on α-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 β-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative α-glucan-processing enzymes.

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Li, Yu Mei, Qiang Li, Sheng Han, Dong Xue Song, Yan Hong Qu, Ting Ting Liu, Yu Xia Zheng, Ying Zi Liu, and Zhi Wen Zhao. "Characterization of Putative a (1,3)-β-D-glucan (curdlan) Synthase for a Low Molecular Weight Curdlan Biosynthesis from Agrobacterium sp. M503." Advanced Materials Research 807-809 (September 2013): 2031–34. http://dx.doi.org/10.4028/www.scientific.net/amr.807-809.2031.

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A β-(1,3)-D-glucan (curdlan) synthase gene for a low molecular weight curdlan biosynthesis, crdSAg, from Agrobacterium sp. M503 was cloned and its encoding protein was characterized by several online protein analysis softwares. The crdSAg consists of 1965-base-pairs Open Reading Frame (ORF) encoding a protein with molecular weight approximate 73.5 kDa, which contains the conserved domain of CESA-CelA_like belonging to glycosyltransferase family 2 (GT2). Moreover, CrdSAg was a membrane protein with seven hydrophobic transmembrance domains. The second structure analysis indicated it was composed of 43.12% α-helix, 17.89% β-sheet, and 38.99% random coil structure. These data will lay a foundation to clarify the biosynthesis mechanism of the low molecular weight curdlan.

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35

Wang, Ping, Cheryl Ingram-Smith, Jill A. Hadley, and Karen J. Miller. "Cloning, Sequencing, and Characterization of thecgmB Gene of Sinorhizobium meliloti Involved in Cyclic β-Glucan Biosynthesis." Journal of Bacteriology 181, no. 15 (August 1, 1999): 4576–83. http://dx.doi.org/10.1128/jb.181.15.4576-4583.1999.

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ABSTRACT Periplasmic cyclic β-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known asRhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic β-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346–6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transformRhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic β-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated thecgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

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Wang, Jie, Lin-Bao Zhu, Yan Ma, Ying-Xue Liu, Hui-Hua Cao, Yu-Ling Wang, Xue Kong, et al. "Bombyx mori β-1,3-Glucan Recognition Protein 4 (BmβGRP4) Could Inhibit the Proliferation of B. mori Nucleopolyhedrovirus through Promoting Apoptosis." Insects 12, no. 8 (August 18, 2021): 743. http://dx.doi.org/10.3390/insects12080743.

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β-1,3-glucan recognition proteins (βGRPs) as pattern recognition receptors (PRRs) play an important role in recognizing various pathogens and trigger complicated signaling pathways in insects. In this study, we identified a Bombyx mori β-1,3-glucan recognition protein gene named BmβGRP4, which showed differential expression, from a previous transcriptome database. The full-length cDNA sequence was 1244 bp, containing an open reading frame (ORF) of 1128 bp encoding 375 amino acids. BmβGRP4 was strongly expressed in the larval stages and highly expressed in the midgut of B. mori larvae in particular. After BmNPV infection, the expression of BmβGRP4 was reduced significantly in the midgut. Furthermore, a significant increase in the copy number of BmNPV was observed after the knockdown of BmβGRP4 in 5th instar larvae, while the overexpression of BmβGRP4 suppressed the proliferation of BmNPV in BmN cells. Subsequently, the expression analysis of several apoptosis-related genes and observation of the apoptosis morphology demonstrated that overexpression of BmβGRP4 facilitated apoptosis induced by BmNPV in BmN cells. Moreover, BmβGRP4 positively regulated the phosphatase and tensin homolog gene (BmPTEN), while expression of the inhibitor of apoptosis gene (BmIAP) was negatively regulated by BmβGRP4. Hence, we hypothesize that BmNPV infection might suppress BmPTEN and facilitate BmIAP to inhibit cell apoptosis by downregulating the expression of BmβGRP4 to escape host antiviral defense. Taken together, these results show that BmβGRP4 may play a role in B. mori response to BmNPV infection and lay a foundation for studying its functions.

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Bozonnet, Sophie, Marguerite Dols-Laffargue, Emeline Fabre, Sandra Pizzut, Magali Remaud-Simeon, Pierre Monsan, and René-Marc Willemot. "Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains." Journal of Bacteriology 184, no. 20 (October 15, 2002): 5753–61. http://dx.doi.org/10.1128/jb.184.20.5753-5761.2002.

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ABSTRACT A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of α-1,6 and α-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and α-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.

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38

Liu, Jun, Huiming Zeng, Xue Li, Lixin Xu, Yingbo Wang, Wei Tang, and Liebao Han. "Isolation and Characterization of the Betaine Aldehyde Dehydrogenase Gene in." Open Biotechnology Journal 4, no. 1 (May 5, 2010): 18–25. http://dx.doi.org/10.2174/1874070701004010018.

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Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the glycine betaine from choline. The BADH gene from turfgrass Ophiopogon japonicus has not been reported. In this study, we first isolated the full length cDNA of betaine aldehyde dehydrogenase gene (OjBADH) from O. japonicus using Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) and Rapid Amplification of cDNA Ends (RACE) techniques. The OjBADH gene (GenBank accession number: DQ645888) has 1785 nucleotides with the 5’ untranscribed region (UTR) of 63 nucleotides, 3’ UTR of 219 nucleotides, and an open reading frame of 1503 nucleotides. This gene encodes a polypeptide of 500 amino acids. It shares a high homology with BADH genes of other Chenopodiaceae species. The putative protein includes a conservative region of phosphofructokinase, aldehyde dehydrogenase, and glutamy phosphoric acid reductase. Overexpression of OjBADH in transgenic tobacco plants demonstrated 2-2.5 folds increase of glycine betaine content and 60- 85% increase of survival rate under salt tolerance. These results suggested that the O. japonicus BADH gene may be used to engineer plants for salt stress tolerance.

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39

Kottom, Theodore J., Charles F. Thomas, and Andrew H. Limper. "Characterization of Pneumocystis carinii PHR1, a pH-Regulated Gene Important for Cell Wall Integrity." Journal of Bacteriology 183, no. 23 (December 1, 2001): 6740–45. http://dx.doi.org/10.1128/jb.183.23.6740-6745.2001.

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ABSTRACT Pneumocystis carinii remains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region codingGSC-1, a gene mediating β-glucan cell wall synthesis in P. carinii. The predicted amino acid sequence of this new gene, termed P. carinii PHR1, exhibited 38% homology to Saccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology to Candida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression of P. carinii PHR1 was examined. As in C. albicans, P. carinii PHR1 expression was repressed under acidic conditions but induced at neutral and more alkaline pH. PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties with P. carinii culture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression of P. carinii genes in related fungi to address functional correlates of P. carinii-encoded proteins. Therefore, the potential role of P. carinii PHR1 in cell wall integrity was examined by assessing its ability to rescue an S. cerevisiae gas1 mutant with absent endogenous Phr1p-like activity. Interestingly, P. carinii PHR1 DNA successfully restored proliferation of S. cerevisiae gas1 mutants under lethal conditions of cell wall stress. These results indicate that P. carinii PHR1encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.

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40

Rixon, J. E., L. M. A. Ferreira, A. J. Durrant, J. I. Laurie, G. P. Hazlewood, and H. J. Gilbert. "Characterization of the gene celD and its encoded product 1,4-β-d-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa." Biochemical Journal 285, no. 3 (August 1, 1992): 947–55. http://dx.doi.org/10.1042/bj2850947.

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A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA constructed in pUC18 and expressed in Escherichia coli was screened for recombinants expressing 4-methylumbelliferyl beta-D-glucoside hydrolysing activity (MUGase). A single MUGase-positive clone was isolated. The MUGase hydrolysed cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose to glucose, by sequentially cleaving glucose residues from the non-reducing end of the cello-oligosaccharides. The Km values for cellobiose and cellohexaose hydrolysis were 1.2 mM and 28 microM respectively. The enzyme exhibited no activity against soluble or insoluble cellulose, xylan and xylobiose. Thus the MUGase is classified as a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) and is designated 1,4-beta-D-glucan glucohydrolase D (CELD). When expressed by E. coli, CELD was located in the cell-envelope fraction; a significant proportion of the native enzyme was also associated with the cell envelope when synthesized by its endogenous host. The nucleotide sequence of the gene, celD, which encodes CELD, revealed an open reading frame of 2607 bp, encoding a protein of M(r) 92,000. The deduced primary structure of CELD was confirmed by the M(r) of CELD (85,000) expressed by E. coli and P. fluorescens subsp. cellulosa, and by the experimentally determined N-terminus of the enzyme purified from E. coli, which showed identity with residues 52-67 of the celD translated sequence. The structure of the N-terminal region of full-length CELD was similar to the signal peptides of P. fluorescens subsp. cellulosa plant-cell-wall hydrolases. Deletion of the N-terminal 47 residues of CELD solubilized MUGase activity in E. coli. CELD exhibited sequence similarity with beta-glucosidase B of Clostridium thermocellum, particularly in the vicinity of the active-site aspartate residue, but did not display structural similarity with the mature forms of cellulases and xylanases expressed by P. fluorescens subsp. cellulosa.

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41

Shimotsuura, Isao, Hiromitsu Kigawa, Motoyasu Ohdera, Howard K. Kuramitsu, and Syozi Nakashima. "Biochemical and Molecular Characterization of a Novel Type of Mutanase from Paenibacillus sp. Strain RM1: Identification of Its Mutan-Binding Domain, Essential for Degradation of Streptococcus mutans Biofilms." Applied and Environmental Microbiology 74, no. 9 (March 7, 2008): 2759–65. http://dx.doi.org/10.1128/aem.02332-07.

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ABSTRACT A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain RM1. The purified enzyme specifically hydrolyzed α-1,3-glucan (mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major etiologic agent in the progression of dental caries, even following brief incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in Escherichia coli demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity whereas the C-terminal domain was responsible for mutanase activity but had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.

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Igarashi, Kiyohiko, Takuya Ishida, Chiaki Hori, and Masahiro Samejima. "Characterization of an Endoglucanase Belonging to a New Subfamily of Glycoside Hydrolase Family 45 of the Basidiomycete Phanerochaete chrysosporium." Applied and Environmental Microbiology 74, no. 18 (August 1, 2008): 5628–34. http://dx.doi.org/10.1128/aem.00812-08.

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ABSTRACT The wood decay fungus Phanerochaete chrysosporium has served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast Pichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (<22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley β-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.

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Zhou, L., G. P. Xue, C. G. Orpin, G. W. Black, H. J. Gilbert, and G. P. Hazlewood. "Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase." Biochemical Journal 297, no. 2 (January 15, 1994): 359–64. http://dx.doi.org/10.1042/bj2970359.

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The cDNA designated celB from the anaerobic rumen fungus Neocallimastix patriciarum contained a single open reading frame of 1422 bp coding for a protein (CelB) of M(r) 53,070. CelB expressed by Escherichia coli harbouring the full-length gene hydrolysed carboxymethylcellulose in the manner of an endoglucanase, but was most active against barley beta-glucan. It also released reducing sugar from xylan and lichenan, but was inactive against crystalline cellulose, laminarin, mannan, galactan and arabinan. The rate of hydrolysis of cellulo-oligosaccharides by CelB increased with increasing chain length from cellotriose to cellopentaose. The predicted structure of CelB contained features indicative of modular structure. The first 360 residues of CelB constituted a fully functional catalytic domain that was homologous with bacterial endoglucanases belonging to cellulase family A, including five which originate from three different species of anaerobic rumen bacteria. Downstream from this domain, and linked to it by a serine/threonine-rich hinge, was a non-catalytic domain containing short tandem repeats, homologous to the C-terminal repeats contained in xylanase A from the same anaerobic fungus. Unlike previous fungal cellulases, genomic celB was devoid of introns. This lack of introns and the homology of its encoded product with rumen bacterial endoglucanases suggest that acquisition of celB by the fungus may at some stage have involved horizontal gene transfer from a prokaryote to N. particiarum.

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44

Terada, Yoshinobu, Kazutoshi Fujii, Takeshi Takaha, and Shigetaka Okada. "Thermus aquaticus ATCC 33923 Amylomaltase Gene Cloning and Expression and Enzyme Characterization: Production of Cycloamylose." Applied and Environmental Microbiology 65, no. 3 (March 1, 1999): 910–15. http://dx.doi.org/10.1128/aem.65.3.910-915.1999.

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ABSTRACT The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of theEscherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coliamylomaltase and the plant D-enzyme.

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45

Zverlov, Vladimir V., Martina Klupp, Jan Krauss, and Wolfgang H. Schwarz. "Mutations in the Scaffoldin Gene, cipA, of Clostridium thermocellum with Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS1447, and Implications for Cellulase Synergism on Crystalline Cellulose." Journal of Bacteriology 190, no. 12 (April 11, 2008): 4321–27. http://dx.doi.org/10.1128/jb.00097-08.

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ABSTRACT Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble β-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

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Murakami, Taira, Tamotsu Kanai, Hiroki Takata, Takashi Kuriki, and Tadayuki Imanaka. "A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1." Journal of Bacteriology 188, no. 16 (August 15, 2006): 5915–24. http://dx.doi.org/10.1128/jb.00390-06.

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ABSTRACT Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the α-1,4 linkage and its subsequent transfer to the α-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain α-glucans, indicating that the reaction product contained many α-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70°C, and thermostability up to 90°C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436ΔH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436ΔH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.

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KIYOHARA, Masashi, Keishi SAKAGUCHI, Kuniko YAMAGUCHI, Toshiyoshi ARAKI, Takashi NAKAMURA, and Makoto ITO. "Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4." Biochemical Journal 388, no. 3 (June 7, 2005): 949–57. http://dx.doi.org/10.1042/bj20050190.

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We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

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48

Iyo, Abiye H., and Cecil W. Forsberg. "Endoglucanase G from Fibrobacter succinogenes S85 belongs to a class of enzymes characterized by a basic C-terminal domain." Canadian Journal of Microbiology 42, no. 9 (September 1, 1996): 934–43. http://dx.doi.org/10.1139/m96-120.

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A 3.6-kb fragment of the Fibrobacter succinogenes S85 DNA was sequenced and found to contain two open reading frames (ORFs) on the same strand separated by 242 nucleotide bases. The translated protein from ORF1 had a predicted mass of 52.3 kDa. In a region of 320 amino acid overlap, it shares a 35% identity with the b-chain of the glutamate synthase of Escherichia coli. The ORF2 protein encodes a 519 residue protein designated CelG. It consists of an ORF of 1557 bp, encoding a polypeptide of 54.5 kDa. The N-terminal region, which contains the catalytic domain, is linked to a C-terminal basic domain, which has a predicted isoelectric point of 10.8. The catalytic domain in endoglucanase G (CelG) is homologous to the family 5 (A) cellulases. The enzyme has an apparent mass of 55 kDa, a pH optimum of 5.5, and temperature optimum of 25 °C. It had a specific activity of 16.5 mmol∙min−1∙mg−1 on barley b-glucan and produced a mixture of cellooligosaccharides from the hydrolysis of acid swollen cellulose and cellooligosaccharides. Antiserum raised against the purified form of CelG in E. coli failed to react with proteins from the native organism when grown on either glucose or crystalline cellulose, but reverse transcription and polymerase chain reaction techniques using RNA from the native organism demonstrated that the celG gene was expressed constitutively. Its distribution amongst subspecies of Fibrobacter was restricted to F. succinogenes S85.Key words: basic terminal domain, Fibrobacter succinogenes, endoglucanase, nucleotide sequence.

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Klockgether, Jens, Oleg Reva, Karen Larbig, and Burkhard Tümmler. "Sequence Analysis of the Mobile Genome Island pKLC102 of Pseudomonas aeruginosa C." Journal of Bacteriology 186, no. 2 (January 15, 2004): 518–34. http://dx.doi.org/10.1128/jb.186.2.518-534.2004.

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ABSTRACT The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with P. aeruginosa clone K chromosomes at one of the two tRNALys genes, pKLC102 is incorporated into the tRNALys gene only close to the pilA locus. Targeting of the other tRNALys copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNAGly-associated genome islands of P. aeruginosa clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements. Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in P. aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.

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Ding, Na, Huihui Cui, Ying Miao, Jun Tang, Qinghe Cao, and Yonghai Luo. "Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato." PeerJ 7 (November 15, 2019): e7933. http://dx.doi.org/10.7717/peerj.7933.

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Background Sweet potato (Ipomoea batatas (L.) Lam.) is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither a complete reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid crop are available, which in turn impedes progress in research studies in I. batatas functional genomics and molecular breeding. Methods In this study, we sequenced full-length transcriptomes in I. batatas and its diploid ancestor I. trifida by single-molecule real-time sequencing and Illumina second-generation sequencing technologies. With the generated datasets, we conducted comprehensive intraspecific and interspecific sequence analyses and experimental characterization. Results A total of 53,861/51,184 high-quality long-read transcripts were obtained, which covered about 10,439/10,452 loci in the I. batatas/I. trifida genome. These datasets enabled us to predict open reading frames successfully in 96.83%/96.82% of transcripts and identify 34,963/33,637 full-length cDNA sequences, 1,401/1,457 transcription factors, 25,315/27,090 simple sequence repeats, 1,656/1,389 long non-coding RNAs, and 5,251/8,901 alternative splicing events. Approximately, 32.34%/38.54% of transcripts and 46.22%/51.18% multi-exon transcripts underwent alternative splicing in I. batatas/I. trifida. Moreover, we validated one alternative splicing event in each of 10 genes and identified tuberous-root-specific expressed isoforms from a starch-branching enzyme, an alpha-glucan phosphorylase, a neutral invertase, and several ABC transporters. Overall, the collection and analysis of large-scale long-read transcripts generated in this study will serve as a valuable resource for the I. batatas research community, which may accelerate the progress in its structural, functional, and comparative genomics studies.

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