title: Characterization of a novel aerobic cellulolytic thermophilic bacterium and its cellulases

authors: Huang, Xiao Ping

abstract: A taxonomic position was established for a thermophilic, cellulolytic bacterium strain COMP.A2 on the basis of both phenotypic and phylogenetic characteristics. The bacterium was a Gram-positive, aerobic endospore-forming rod. Growth was observed between 60 and 70°C, with an optimum at 65°C. The optimum pH for growth was between 6.5 and 7.5. A wide range of substrates including Avicel and Sigmacell 20 could be utilised as sole carbon and energy source at 70°C. Lactate and acetate were produced when grown on cellobiose or amorphous cellulose medium. The organism was very sensitive to antibiotics such as penicilin G, streptomycin, and neomycin. Growth was inhibited by low concentrations of agar or agarose. A preference for uptake of cellobiose over glucose as the substrate was observed in a mixture of glucose and cellobiose medium. The bacterium was unable to grow autotrophically using hydrogen and carbon dioxide. The lack of alicyclic fatty acid in the cell membrane and the neutral growth pH indicates that the strain is not a member of the genus Alicyclobacillus. The G+C content of the DNA was 62.4 mol%. The 16S rRNA sequences showed that strain COMP.A2 is phylogenetically distinct from other members of genus Bacillus and forms a cluster with the genus Alicylobacillus and two facultatively chemolithoautotrophilic hydrogen-oxidizing Bacillus species, B. tusciae and B. schlegelii. Strain COMP.A2 is tentatively proposed as a new species of a new genus, namely Caldibacillus cellulovorans gen. nov., sp. nov. on the basis of its physiological and phylogenetic characters.

Two CMCases (CMCase A and CMCase B) and one β-glucosidase have been purified from the culture filtrate of strain COMP.A2 by Phenyl-Sepharose chromatography, ion exchange chromatography and chromatofocusing. CMCase A and β-glucosidase were shown to be electrophoretically homogeneous on PAGE and SDS-PAGE. CMCase B was partially purified.

The molecular weight of CMCase A was determined to be 85.1 kDa by SDS-PAGE and 174 kDa by size-exclusion chromatography. The isoelectric point of CMCase A was 4.12. With an assay time of 10 min, CMCase A displayed highest activity at 80°C. An optimum pH for activity was in the range of pH 6.5 to 7.0. An active energy was 46.6 KJ mol⁻¹. The enzyme did not lose activity over one hour at 70°C and still possessed 83% of the original activity after three hours incubation at 70°C. The half-life at 80°C was 32 min and the thermostability was increased by the addition of bovine serum albumin. The enzyme activity was inhibited by heavy metal ions (Zn²⁺ and Hg²⁺) and the thiol-specific inhibitor p-chloromercuribenzenesulphonic acid (pCMPS). The CMCase A showed greatest hydrolytic activity on CMC and low activity on Avicel and xylan. CMCase A hydrolyses CMC or amorphous cellulose in an exo-model of action following initial and random attack. The Km value of the enzyme for CMC was 3.4 mg ml⁻¹. The enzyme cleaved cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. Cellotetraose was cleaved to cellobiose. Cellotriose and cellobiose were not degraded.

The β-glucosidase was largely cell-associated. The enzyme has a molecular weight of 52.5 kDa as determin by SDS-PAGE and an isoelectric point of 4.43. With an assay time of 15 min, β-glucosidase displayed highest activity at 75°C. The enzyme showed optimum activity in the range of pH 5.5 to 7.0. An active energy was 35.4 KJ mol⁻¹. The enzyme was inhibited by the thiol-specific inhibitor pCMPS and heavy metal ions (Cd²⁺, Hg²⁺, Ag⁺). The thermostability of β-glucosidase was increased by the addition of trehalose. The half-life of β-glucosidase was less than 5 minutes at 80°C and 2.5 hours at 70°C in comparison with 24 minutes at 80°C and 11.75 hours at 70°C in the presence of trehalose. The enzyme was competitive inhibited by gluconolactone. The β-glucosidase showed very broad substrate specificity and was active against sophorose, cellobiose and pNPG with Km values of 2.0 mM, 3.4 mM and 0.36mM, respectively.

text: Characterization of a novel aerobic cellulolytic thermophilic bacterium and its cellulases A taxonomic position was established for a thermophilic, cellulolytic bacterium strain COMP.A2 on the basis of both phenotypic and phylogenetic characteristics. The bacterium was a Gram-positive, aerobic endospore-forming rod. Growth was observed between 60 and 70°C, with an optimum at 65°C. The optimum pH for growth was between 6.5 and 7.5. A wide range of substrates including Avicel and Sigmacell 20 could be utilised as sole carbon and energy source at 70°C. Lactate and acetate were produced when grown on cellobiose or amorphous cellulose medium. The organism was very sensitive to antibiotics such as penicilin G, streptomycin, and neomycin. Growth was inhibited by low concentrations of agar or agarose. A preference for uptake of cellobiose over glucose as the substrate was observed in a mixture of glucose and cellobiose medium. The bacterium was unable to grow autotrophically using hydrogen and carbon dioxide. The lack of alicyclic fatty acid in the cell membrane and the neutral growth pH indicates that the strain is not a member of the genus Alicyclobacillus. The G+C content of the DNA was 62.4 mol%. The 16S rRNA sequences showed that strain COMP.A2 is phylogenetically distinct from other members of genus Bacillus and forms a cluster with the genus Alicylobacillus and two facultatively chemolithoautotrophilic hydrogen-oxidizing Bacillus species, B. tusciae and B. schlegelii. Strain COMP.A2 is tentatively proposed as a new species of a new genus, namely Caldibacillus cellulovorans gen. nov., sp. nov. on the basis of its physiological and phylogenetic characters.

Two CMCases (CMCase A and CMCase B) and one β-glucosidase have been purified from the culture filtrate of strain COMP.A2 by Phenyl-Sepharose chromatography, ion exchange chromatography and chromatofocusing. CMCase A and β-glucosidase were shown to be electrophoretically homogeneous on PAGE and SDS-PAGE. CMCase B was partially purified.

The molecular weight of CMCase A was determined to be 85.1 kDa by SDS-PAGE and 174 kDa by size-exclusion chromatography. The isoelectric point of CMCase A was 4.12. With an assay time of 10 min, CMCase A displayed highest activity at 80°C. An optimum pH for activity was in the range of pH 6.5 to 7.0. An active energy was 46.6 KJ mol⁻¹. The enzyme did not lose activity over one hour at 70°C and still possessed 83% of the original activity after three hours incubation at 70°C. The half-life at 80°C was 32 min and the thermostability was increased by the addition of bovine serum albumin. The enzyme activity was inhibited by heavy metal ions (Zn²⁺ and Hg²⁺) and the thiol-specific inhibitor p-chloromercuribenzenesulphonic acid (pCMPS). The CMCase A showed greatest hydrolytic activity on CMC and low activity on Avicel and xylan. CMCase A hydrolyses CMC or amorphous cellulose in an exo-model of action following initial and random attack. The Km value of the enzyme for CMC was 3.4 mg ml⁻¹. The enzyme cleaved cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. Cellotetraose was cleaved to cellobiose. Cellotriose and cellobiose were not degraded.

The β-glucosidase was largely cell-associated. The enzyme has a molecular weight of 52.5 kDa as determin by SDS-PAGE and an isoelectric point of 4.43. With an assay time of 15 min, β-glucosidase displayed highest activity at 75°C. The enzyme showed optimum activity in the range of pH 5.5 to 7.0. An active energy was 35.4 KJ mol⁻¹. The enzyme was inhibited by the thiol-specific inhibitor pCMPS and heavy metal ions (Cd²⁺, Hg²⁺, Ag⁺). The thermostability of β-glucosidase was increased by the addition of trehalose. The half-life of β-glucosidase was less than 5 minutes at 80°C and 2.5 hours at 70°C in comparison with 24 minutes at 80°C and 11.75 hours at 70°C in the presence of trehalose. The enzyme was competitive inhibited by gluconolactone. The β-glucosidase showed very broad substrate specificity and was active against sophorose, cellobiose and pNPG with Km values of 2.0 mM, 3.4 mM and 0.36mM, respectively.

year: 1999

title: Cylindrical spikes

authors: Moughal, Muhammad Zubair Ali

subjects: Science & Technology

abstract: The Geroch/Stephani transformation is a solution-generating transformation, and may generate spiky solutions. The spikes in solutions generated so far are either early-time permanent spikes or transient spikes. We want to generate a solution with a late-time permanent spike. We achieve this by applying Stephani's transformation with the rotational Killing vector field (KVF) of the locally rotationally symmetric Jacobs solution. The late-time permanent spike occurs along the cylindrical axis. Using a mixed KVF, the generated solution also features a rich variety of transient structures. We introduce a new technique to analyse these structures. Our findings lead us to discover a transient behaviour, which we call the overshoot transition. These discoveries compel us to revise the description of transient spikes.

text: Cylindrical spikes The Geroch/Stephani transformation is a solution-generating transformation, and may generate spiky solutions. The spikes in solutions generated so far are either early-time permanent spikes or transient spikes. We want to generate a solution with a late-time permanent spike. We achieve this by applying Stephani's transformation with the rotational Killing vector field (KVF) of the locally rotationally symmetric Jacobs solution. The late-time permanent spike occurs along the cylindrical axis. Using a mixed KVF, the generated solution also features a rich variety of transient structures. We introduce a new technique to analyse these structures. Our findings lead us to discover a transient behaviour, which we call the overshoot transition. These discoveries compel us to revise the description of transient spikes.

year: 2021

title: Testing a suicidal ideation-to-action framework among Queer and Takatāpui people in Aotearoa New Zealand: An examination of the three-step theory

authors: Schimanski, Irie

abstract: The three-step theory (3ST) of suicide is well-established in the general population and shows promise for explaining suicidal ideation among Queer samples. This study examined the applicability of the 3ST using data from 250 Queer (n = 213) and Takatāpui (n = 37) people aged 18-74 living in Aotearoa New Zealand. The analytic approach entailed hierarchical multiple regression, ANOVA, t-tests, and logistic regression. Step-one was partially supported, with discrimination and hopelessness independently associated with suicidal ideation, but not their interaction. Step-two was fully supported, showing social support associated with lower suicidal ideation when social support exceeded discrimination, compared with the inverse. Full support for step-three was observed, finding higher self-harm among participants with histories of attempted suicide than those who had never attempted. An adapted version of the 3ST for Queer and Takatāpui people is presented, offering novel approaches to testing the ideation-to-action framework with these communities.

text: Testing a suicidal ideation-to-action framework among Queer and Takatāpui people in Aotearoa New Zealand: An examination of the three-step theory The three-step theory (3ST) of suicide is well-established in the general population and shows promise for explaining suicidal ideation among Queer samples. This study examined the applicability of the 3ST using data from 250 Queer (n = 213) and Takatāpui (n = 37) people aged 18-74 living in Aotearoa New Zealand. The analytic approach entailed hierarchical multiple regression, ANOVA, t-tests, and logistic regression. Step-one was partially supported, with discrimination and hopelessness independently associated with suicidal ideation, but not their interaction. Step-two was fully supported, showing social support associated with lower suicidal ideation when social support exceeded discrimination, compared with the inverse. Full support for step-three was observed, finding higher self-harm among participants with histories of attempted suicide than those who had never attempted. An adapted version of the 3ST for Queer and Takatāpui people is presented, offering novel approaches to testing the ideation-to-action framework with these communities.

year: 2025-08-01