Effect of Yeast Extract and Vitamin B(12) on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B(12), biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B(12) alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Advances in development of a genetic system for Thermoanaerobacterium spp.: expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.     

Studies on the anaerobic degradation of crystalline cellulose by Clostridium thermocellum using a new assay

Abstract The anaerobic degradation of microcrystalline cellulose by thermostable cellulolytic enzyme complexes from Clostridium thermocellum JW20 (ATCC 31449) was monitored. For quantitative investigations as enzyme-coupled spectrophotometric assay has been developed. The assay allows for the evaluation of the release of cellubiose-/glucose-units from native cellulose. Kinetic studies revealed that the anaerobic breakdown of crystalline cellulose (CC) at 60°C follows Michaelis-Menten kinetics K _m CC values have been determined for different aggregation states of the cellulolytic complex. The presented assay seems well suited to screen for CC-degrading enzymes of various sources, and to further explore the mechanism of CC-breakdown.     

Localization and immunological characterization of the cellulolytic enzyme system in Clostridium thermocellum

Abstract The cellulolytic enzyme complex from Clostridium thermocellum JW 20 was purified from the cellulose to which the enzyme was bound during growth. After centrifugation and gel filtration the enzyme complex was analyzed by SDS-PAGE. Three subunits with apparent molecular weights of 195 000 Da, 97 000 Da and 72 000 Da were purified by preparative SDS-PAGE and electroelution. Polyclonal antibodies directed against these three subunits were raised in rabbits. The specificity of the antisera was tested with immunochemical methods. Cross reactions with other subunits of the cellulase complex were observed. Immunoelectron microscopy of protein-A gold labeled, resin embedded cells indicated that the three types of subunits were located in the outer region of the cytoplasm and on structures at the outside of the cell wall.     

Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.     

Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates

Interest in solid substrate cultivation (SSC) techniques is gaining for biochemical production from renewable resources; however, heat and mass transfer problems may limit application of this technique. The use of anaerobic thermophiles in SSC offers a unique solution to overcoming these challenges. The production potential of nine thermophilic anaerobic bacteria was examined on corn stover, sugar cane bagasse, paper pulp sludge, and wheat bran in submerged liquid cultivation (SmC) and SSC. Production of acetate, ethanol, and lactate was measured over a 10 day period, and total product concentrations were used to compare the performance of different organism-substrate combinations using the two cultivation methods. Overall microbial activity in SmC and SSC was dependent on the organism and growth substrate. Clostridium thermocellum strains JW20, LQRI, and 27405 performed significantly better in SSC when grown on sugar cane bagasse and paper pulp sludge, producing at least 70 and 170 mM of total products, respectively. Growth of C. thermocellum strains in SSC on paper pulp sludge proved to be most favorable, generating at least twice the concentration of total products produced in SmC (p-value < 0.05). Clostridium thermolacticum TC21 demonstrated growth on all substrates producing 30-80 and 60-116 mM of total product in SmC and SSC, respectively. Bacterial species with optimal growth temperatures of 70 degrees C grew best on wheat bran in SmC, producing total product concentrations of 45-75 mM. For some of the organism-substrate combinations total end product concentrations in SSC exceeded those in SmC, indicating that SSC may be a promising alternative for microbial activity and value-added biochemical production.     

Comparison of the cellulolytic systems ofClostridium thermocellum YM4 and JW20

Summary The hyper-cellulolytic strain ofClostridium thermcellum, YM4, produced about 20-fold more CMCase than did strain JW20 (ATCC 31449). Most part of the difference between their enzyme production resided in the enzyme fraction which was free in the culture fluid but could bind to cellulose. The chief distinction of the cellulolytic system of strain YM4 from that of JW20 was the lack of the polycellulosome.     

Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis cell envelopes

Binding parameters were determined for the SLH (S-layer homologous) domains from the Clostridium thermocellum outer layer protein OlpB, from the C. thermocellum S-layer protein SlpA, and from the Bacillus anthracis S-layer proteins EA1 and Sap, using cell walls from C. thermocellum and B. anthracis. Each SLH domain bound to C. thermocellum and B. anthracis cell walls with a different KD, ranging between 7.1 x 10(-7) and 1.8 x 10(-8) M. Cell wall binding sites for SLH domains displayed different binding specificities in C. thermocellum and B. anthracis. SLH-binding sites were not detected in cell walls of Bacillus subtilis. Cell walls of C. thermocellum lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from C. thermocellum cells by sodium dodecyl sulfate treatment, bound the SLH domains from C. thermocellum but not those from B. anthracis proteins. A corresponding component was not found in B. anthracis.     

Electrically enhanced ethanol fermentation by <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ethanol production by Clostridium thermocellum ATCC 35609 and Saccharomyces cerevisiae ATCC 26603 was improved in an electrochemical bioreactor system. It was increased by 61% with Cl. thermocellum and 12% with S. cerevisiae in the presence of -1.5 V of electric potential. These increases were attributed to high production rates due to regeneration and availability of increased reduced equivalents in the presence of electric potential. The electric current caused considerable shift in the metabolite concentrations on a molar basis in Cl. thermocellum fermentation but less in S. cerevisiae fermentation. Increasing electric potential in Cl. thermocellum fermentation resulted in less acetate and more lactate production. Acetate production was also reduced with increased electric potential in S. cerevisiae fermentation. The high electric potential of -5 V adversely affected the Cl. thermocellum fermentation, but not the S. cerevisiae fermentation even at a high electric potential of -10 V.     

Comparison of Bacteriocins Produced by Lactic-Acid Bacteria Isolated from Boza, a Cereal-Based Fermented Beverage from the Balkan Peninsula

Boza is a low-pH and low-alcohol cereal-based beverage produced in the Balkan Peninsula. From a total population of 9 × 10⁶ colony-forming units ml⁻¹, four isolates (JW3BZ, JW6BZ, JW11BZ, and JW15BZ) produced bacteriocins active against a broad spectrum of Gram-positive bacteria. Bacteriocin JW15BZ inhibited the growth of Klebsiella pneumoniae. The producer strains were identified as Lactobacillus plantarum (strains JW3BZ and JW6BZ) and L. fermentum (strains JW11BZ and JW15BZ). The spectrum of antimicrobial activity, characteristics, and mode of action of these bacteriocins were compared with bacteriocins previously described for lactic-acid bacteria isolated from boza.     

WHBE兔血液蛋白的特性

目的比较大耳白黑眼兔（WHBE兔）与日本大耳白兔（JW兔）和新西澜兔（NZW兔）血液蛋白的多态性,从分子水平了解WHBE兔特殊性状的遗传背景。方法采用聚丙烯酰胺凝胶电泳对WHBE兔、JW兔和NZW兔血液中的前白蛋白（Pr）,后白蛋白（Po）,前转铁蛋白1（Prt1）,前转铁蛋白2（Prt2）,转铁蛋白1（Tf1）,转铁蛋白2（Tf2）,后转铁蛋白（Ptf）,慢α球蛋白（Sag）、血红蛋白（Hbα、Hbβ）和白蛋白（Alb）共11个蛋白位点进行检测。结果Pr、Ptf、Hbα、Hbβ、Alb、Po、Sag和Prt2在所有实验兔个体中的表型一致,而Tf1、Tf2和Prt1的表型在各实验兔品系间存在显著差异。在Tf1位点,NZW兔以BC表型为主,无AA型;JW兔只有AA型,而WHBE兔以AB型为主。在Tf2位点,NZW兔有AA型,而JW兔和WHBE兔无AA型。在Prt1位点,NZW兔无AB、BB和BC表型而有AC表型。NZW兔蛋白位点的平均杂合度大于JW兔和WHBE兔。JW兔与WHBE兔的遗传距离最近。结论Tf1、Tf2和Prt1可作为区分NZW兔、JW兔和WHBE兔的特征标记,并且与WHBE兔特殊性状形成有关。JW兔与WHBE兔的亲缘关系最近。     

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.     

Comparison of alcohol dehydrogenases from wild-type and mutant strain,JW200 Fe 4, of ‘Thermoanaerobacter ethanolicus’

Summary A mutant strain of Thermoanaerobacter ethanolicus (ATCC 31 550) designated JW200 Fe 4 contains primary and secondary alcohol dehydrogenases (ADHs). The primary ADH from JW000 Fe 4 was formed early in the growth cycle compared to the primary ADH form the wild-type strain (JW200 wt). The secondary ADH displayed 2.5-fold greater activity during the growth cycle of JW200 Fe 4 compared to the secondary ADH form JW200 wt. Both primary and secondary ADHs from JW200 Fe 4 were purified to homogeneity ADHs from JW200 Fe 4 were purified to homogeneity as determined by sodium dodecyl sulphate-gel electrophoresis. Relative molecular weight estimations indicated that both ADHs were tetrameric. Each ADH from JW200 Fe 4 contained approximately four Zn atoms per subunit and displayed Arrhenius plots similar to the ADHs from JW200 wt. The substrate specificity for the ADHs from JW200 Fe 4 was similar to that of the ADHs from JW200 wt. The secondary ADH oxidized 2-propanol at 51 times the rate of ethanol. Both ADHs from JW200 Fe 4 apparently reduce acetaldehyde to ethanol while only the secondary ADH from JW200 wt was suggested to contribute significantly to ethanol production.     

Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase

In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.     

记吉林集安仙人洞的鹿类化石,兼述我国斑鹿化石的分类

本文记述了产于吉林省集安县大路乡仙人洞洞穴堆积中的晚更新世鹿超科化石。这些化石可以分别归入麝科Moschidae和鹿科Cervidae,共四个种:Moschus moschiferus, Cervus (Sika) nippon horiulorum, Megaloceros ordosianus和Capreolus manchuricus。它们代表四种大小不同的鹿类动物,同为我国北方猛犸象-披毛犀动物群的成员。本文还指出斑鹿亚属的拉丁学名应为Sika,而不是Pseudaxis。产于我国的斑鹿化石可分为四个种。     

Advances in Development of a Genetic System for Thermoanaerobacterium spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.     

Chronic hypertriglyceridemia in young watanabe heritable hyperlipidemic rabbits impairs endothelial and medial smooth muscle function

Several studies have suggested that hypertriglyceridemia is a common risk factor for coronary heart disease. Although increasing serum levels of triglyceride correlate with hypercoagulability, little is known about the contribution of hypertriglyceridemia to vascular function. We successfully segregated two lines of rabbits with genetically-determined severely high (TGH; 2764 +/- 413 mg/dl) and moderately high (TGL; 191 +/- 12 mg/dl) levels of triglyceride, but with comparable levels of total cholesterol, from Watanabe heritable hyperlipidemic rabbits. To determine whether hypertriglyceridemia was involved in alterations of vascular function, we conducted isometric tension studies and analyzed protein expression on thoracic aortic rings isolated from young (3-4 month) TGH, TGL and Japanese White rabbit (JW). No difference in percentage of plaque area in the thoracic aorta was found between TGH and TGL. Relaxing responses, evoked by sodium nitroprusside were similar in JW, TGL and TGH, but endothelium-dependent relaxation to acetylcholine was impaired in TGH compared with JW or TGL (maximal relaxation in JW; 83.5 +/- 2.7%, TGL; 79.9 +/- 5.3%, TGH; 59.1 +/- 5.7%, p<0.05). Relaxation to A23187 was also attenuated in TGH compared with JW, but not significantly different between TGL and JW. Endothelium-independent relaxation elicited by isoproterenol in TGH was significantly decreased compared with JW or TGL (maximal relaxation in JW; 95.2 +/- 2.6% TGL; 91.0 +/- 4.9%, TGH; 75.1 +/- 5.2%, p<0.05). Protein expression of angiotensin II type-1 receptor was increased in TGH and that of nitric oxide synthases-3 was attenuated in TGH compared with TGL. This is the first study showing that endothelium-dependent and -independent vascular relaxation under the condition of combined hyperlipidemia was severely impaired as compared to that under only hypercholesterolemia. These results suggest that hypertriglyceridemia aggravates functional impairment induced by hypercholesterolemia in endothelial and smooth muscle cells.     

Cohesin-dockerin interactions within and between Clostridium josui and Clostridium thermocellum: binding selectivity between cognate dockerin and cohesin domains and species specificity

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.