Botrytis cinerea hypovirulent strain △BcSpd1 induced Panax ginseng defense.

Background: Gray mold, caused by Botrytis cinerea, is one of the major fungal diseases in agriculture. Biological methods are preferred over chemical fungicides to control gray mold since they are less toxic to the environment and could induce the resistance to pathogens in plants. In this work, we try to understand if ginseng defense to B. cinerea could be induced by fungal hypovirulent strain △BcSpd1. BcSpd1 encodes Zn(II)2Cys6 transcription factor which regulates fungal pathogenicity and we recently reported △BcSpd1 mutants reduced fungal virulence. Methods: We performed transcriptomic analysis of the host to investigate the induced defense response of ginseng treated by B. cinerea △BcSpd1. The metabolites in ginseng flavonoids pathway were determined by UPLC-ESI-MS/MS and the antifungal activates were then performed. Results: We found that △BcSpd1 enhanced the ginseng defense response when applied to healthy ginseng leaves and further changed the metabolism of flavonoids. Compared with untreated plants, the application of △BcSpd1 on ginseng leaves significantly increased the accumulation of p-coumaric acid and myricetin, which could inhibit the fungal growth. Conclusion: B. cinerea △BcSpd1 could effectively induce the medicinal plant defense and is referred to as the biological control agent in ginseng disease management.

Transcriptome and metabolite analyses indicated the underlying molecular responses of Asian ginseng (Panax ginseng) toward Colletotrichum panacicola infection.

Panax ginseng Meyer is one of the most valuable plants and is widely used in China, while ginseng anthracnose is one of the most destructive diseases. Colletotrichum panacicola could infect ginseng leaves and stems and causes serious anthracnose disease, but its mechanism is still unknown. Here, transcriptome and metabolism analyses of the host leaves were conducted to investigate the ginseng defense response affected by C. panacicola. Upon C. panacicola infection, ginseng transcripts altered from 14 to 24 h, and the expression of many defense-related genes switched from induction to repression. Consequently, ginseng metabolites in the flavonoid pathway were changed. Particularly, C. panacicola repressed plant biosynthesis of the epicatechin and naringin while inducing plant biosynthesis of glycitin, vitexin/isovitexin, and luteolin-7-O-glucoside. This work indicates C. panacicola successful infection of P. ginseng by intervening in the transcripts of defense-related genes and manipulating the biosynthesis of secondary metabolites, which might have antifungal activities.

Tea plant (Camellia sinensis) lipid metabolism pathway modulated by tea field microbe (Colletotrichum camelliae) to promote disease.

Tea is one of the most popular healthy and non-alcoholic beverages worldwide. Tea anthracnose is a disease in tea mature leaves and ultimately affects yield and quality. Colletotrichum camelliae is a dominant fungal pathogen in the tea field that infects tea plants in China. The pathogenic factors of fungus and the susceptible factors in the tea plant are not known. In this work, we performed molecular and genetic studies to observe a cerato-platanin protein CcCp1 from C. camelliae, which played a key role in fungal pathogenicity. △CcCp1 mutants lost fungal virulence and reduced the ability to produce conidia. Transcriptome and metabolome were then performed and analysed in tea-susceptible and tea-resistant cultivars, Longjing 43 and Zhongcha 108, upon C. camelliae wild-type CCA and △CcCp1 infection, respectively. The differentially expressed genes and the differentially accumulated metabolites in tea plants were clearly overrepresented such as linolenic acid and linoleic acid metabolism, glycerophospholipid metabolism, phenylalanine biosynthesis and metabolism, biosynthesis of flavonoid, flavone and flavonol etc. In particular, the accumulation of jasmonic acid was significantly increased in the susceptible cultivar Longjing 43 upon CCA infection, in the fungal CcCp1 protein dependent manner, suggesting the compound involved in regulating fungal infection. In addition, other metabolites in the glycerophospholipid and phenylalanine pathway were observed in the resistant cultivar Zhongcha 108 upon fungal treatment, suggesting their potential role in defense response. Taken together, this work indicated C. camelliae CcCp1 affected the tea plant lipid metabolism pathway to promote disease while the lost function of CcCp1 mutants altered the fungal virulence and plant response.

The necrotroph Botrytis cinerea promotes disease development in Panax ginseng by manipulating plant defense signals and antifungal metabolites degradation.

Background: Panax ginseng Meyer is one of the most valuable medicinal plants which is enriched in anti-microbe secondary metabolites and widely used in traditional medicine. Botrytis cinerea is a necrotrophic fungus that causes gray mold disease in a broad range of hosts. B. cinerea could overcome the ginseng defense and cause serious leaf and root diseases with unknown mechanism. Methods: We conducted simultaneous transcriptomic and metabolomic analysis of the host to investigate the defense response of ginseng affected by B. cinerea. The gene deletion and replacement were then performed to study the pathogenic gene in B. cinerea during ginseng - fungi interaction. Results: Upon B. cinerea infection, ginseng defense responses were switched from the activation to repression, thus the expression of many defense genes decreased and the biosynthesis of antifungal metabolites were reduced. Particularly, ginseng metabolites like kaempferol, quercetin and luteolin which could inhibit fungi growth were decreased after B. cinerea infection. B. cinerea quercetin dioxygenase (Qdo) involved in catalyzing flavonoids degradation and △BcQdo mutants showed increased substrates accumulation and reduced disease development. Conclusion: This work indicates the flavonoids play a role in ginseng defense and BcQdo involves in B. cinerea virulence towards the P. ginseng. B. cinerea promotes disease development in ginseng by suppressing of defense related genes expression and reduction of antifungal metabolites biosynthesis.

The Necrotroph Botrytis cinerea BcSpd1 Plays a Key Role in Modulating Both Fungal Pathogenic Factors and Plant Disease Development.

Botrytis cinerea is a necrotrophic microbe that causes gray mold disease in a broad range of hosts. In the present study, we conducted molecular microbiology and transcriptomic analyses of the host-B. cinerea interaction to investigate the plant defense response and fungal pathogenicity. Upon B. cinerea infection, plant defense responses changed from activation to repression; thus, the expression of many defense genes decreased in Arabidopsis thaliana. B. cinerea Zn(II)2Cys6 transcription factor BcSpd1 was involved in the suppression of plant defense as ΔBcSpd1 altered wild-type B05.10 virulence by recovering part of the defense responses at the early infection stage. BcSpd1 affected genes involved in the fungal sclerotium development, infection cushion formation, biosynthesis of melanin, and change in environmental pH values, which were reported to influence fungal virulence. Specifically, BcSpd1 bound to the promoter of the gene encoding quercetin dioxygenase (BcQdo) and positively affected the gene expression, which was involved in catalyzing antifungal flavonoid degradation. This study indicates BcSpd1 plays a key role in the necrotrophic microbe B. cinerea virulence toward plants by regulating pathogenicity-related compounds and thereby suppressing early plant defense.

Urate oxidase from tea microbe Colletotrichum camelliae is involved in the caffeine metabolism pathway and plays a role in fungal virulence.

Tea is one of the most well-known, healthy beverages in the world. Tea plants produce caffeine as a secondary metabolite. Colletotrichum camelliae is one of the most important microbes frequently isolated from tea fields, and it causes anthracnose disease in tea plant. In the present work, we performed molecular microbiology and transcriptomic analyses of the C. camelliae - tea plant interaction to investigate the mechanism of fungal virulence and plant defense. Upon infection of tea plant with C. camelliae, we observed alterations in the expression of fungal transcripts, including those of many genes associated with caffeine metabolism, such as those encoding various transporters, xanthine dehydrogenase, and urate oxidase (UOX). In particular, the deletion of C. camelliae urate oxidase (CcUOX), which is involved in the caffeine metabolism pathway, reduced fungal tolerance to caffeine, and impaired fungal virulence. CcUOX is involved in caffeine metabolism by the degradation of uric acid contents. C. camelliaeΔCcUOX mutants impaired uric acid degradation in vivo. The CcUOX gene was cloned from C. camelliae, overexpressed in Escherichia coli, and the recombinant CcUOX protein displayed maximum activity at 30°C and a pH of 4.0. The recombinant CcUOX efficiently reduced uric acid in vitro suggesting a promising application in caffeine-contaminated environment management and in producing food with low purine contents to prevent uric acid related human diseases, such as hyperuricemia and gout.

Effects of a Novel Rotator Cuff Rehabilitation Device on Shoulder Strength and Function.

ABSTRACT: Savitzky, JA, Abrams, LR, Galluzzo, NA, Ostrow, SP, Protosow, TJ, Liu, SA, Handrakis, JP, and Friel, K. Effects of a novel rotator cuff rehabilitation device on shoulder strength and function. J Strength Cond Res 35(12): 3355-3363, 2021-The glenohumeral joint, a multiaxial ball and socket joint, has inherent instability counterbalanced by the muscular stability of the rotator cuff (RC) and connective tissue. Exercise has been shown to alleviate pain and disability arising from degenerative changes of the RC due to overuse, trauma, or poor posture. This study compared the training effects of ShoulderSphere (SS), an innovative device that uses resistance to centrifugal force, to TheraBand (TB), a traditional device that uses resistance to elasticity. Thirty-five healthy male and female adults (24.2 ± 2.4 years) were randomized into 3 groups: SS, TB, and control. Five outcomes were assessed before and after the twice-weekly, 6-week intervention phase: strength (shoulder flexion [Fx], extension [Ext], external rotation [ER], and internal rotation [IR]), proprioception (6 positions), posterior shoulder endurance (ShEnd), stability (Upper Quarter Y-Balance Test [YBal] (superolateral [YBalSup], medial [YBalMed], and inferolateral [YBalInf]), and power (seated shot put [ShtPt]). Data were analyzed using a 3 (group: SS, TB, and control) × 2 (time: pre and post) generalized estimating equation. Analyses demonstrated a main effect of time for all strength motions (p < 0.01): YBalInf (p < 0.0001), ShtPt (p < 0.05), and ShEnd (p < 0.0001) but no interaction effects of group × time. There were no main or interaction effects for proprioception. Both SS and TB groups had significant within-group increases in Ext, IR, YBalInf, and ShEnd. Only the SS group had significant increases in ER, Fx, and ShtPt. ShoulderSphere demonstrated comparable conditioning effects with TB and may afford additional strength gains in Fx and ER, and power. ShoulderSphere should be considered a viable alternative in RC conditioning.

Development of a DNA-based real-time PCR assay for the quantification of Colletotrichum camelliae growth in tea (Camellia sinensis).

BACKGROUND: Tea, which is produced from new shoots of existing tea plants (Camellia sinensis), is one of the most popular, non-alcoholic, healthy beverages worldwide. Colletotrichum camelliae is one of the dominant fungal pathogens of tea. The interaction of C. camelliae with tea could be a useful pathosystem to elucidate various aspects of woody, medicinal plant-fungal interactions. Currently, many studies characterizing resistance or virulence and aggressiveness use lesion size at the infection sites on the leaves to quantify the growth of the pathogen. However, this method does not offer the sensitivity needed for the robust quantification of small changes in aggressiveness or the accurate quantification of pathogen growth at the early stages of infection. RESULTS: A quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed for the quantification of C. camelliae growth on tea plant. This method was based on the comparison of fungal DNA in relation to plant biomass. This assay was used to investigate the phenotypes of tea plant cultivars in response to C. camelliae infection. Two cultivars, Zhongcha 108 (ZC108) and Longjing 43 (LJ43), were tested with this method. ZC108 was previously reported as an anthracnose-resistant cultivar against C. camelliae, while LJ43 was susceptible. The traditional lesion measurement method showed that both cultivars were susceptible to a virulent strain of C. camelliae, while the qRT-PCR approach indicated that very little fungal growth occurred in the anthracnose-resistant cultivar ZC108. The observed results in this study were consistent with previously published research. In addition, the DNA-based real-time PCR method was applied for analysis of pathogenic differences in general C. camelliae isolates and among several Colletotrichum spp that infect tea. CONCLUSIONS: This study showed that the DNA-based qRT-PCR technique is rapid, highly sensitive and easily applicable for routine experiments and could be used in screening for resistant tea plant cultivars or to identify differences in pathogen aggressiveness within and among Colletotrichum species.

Validation of Reliable Reference Genes for RT-qPCR Studies of Target Gene Expression in Colletotrichum camelliae During Spore Germination and Mycelial Growth and Interaction With Host Plants.

The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. Colletotrichum camelliae Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of C. camelliae, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in C. camelliae thus far. In this study, 12 candidate genes (i.e., CcSPAC6B12.04c, CcWDR83, Cchp11, Ccnew1, CcHplo, CcRNF5, CcHpcob, CcfaeB-2, CcYER010C, CcRNM1, CcUP18, and CcACT) were isolated from C. camelliae and assessed as potential reference genes. The expression stability of these genes in C. camelliae during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that Cenew1, CcHplo, and CcSPAC6B12.04c were the most stable reference genes in C. camelliae under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of C. camelliae.

The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications.

In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.

Botrytis cinerea B05.10 promotes disease development in Arabidopsis by suppressing WRKY33-mediated host immunity.

The large WRKY transcription factor family is mainly involved in regulating plant immune responses. Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic processes towards Botrytis cinerea strain 2100 infection and is essential for resistance. In contrast to B. cinerea strain 2100, the strain B05.10 is virulent on wild-type (WT) Col-0 Arabidopsis plants highlighting the genetic diversity within this pathogen species. We analysed how early WRKY33-dependent responses are affected upon infection with strain B05.10 and found that most of these responses were strongly dampened during this interaction. Ectopic expression of WRKY33 resulted in complete resistance towards this strain indicating that virulence of B05.10, at least partly, depends on suppressing WRKY33 expression/protein accumulation. As a consequence, the expression levels of direct WRKY33 target genes, including those involved in the biosynthesis of camalexin, were also reduced upon infection. Concomitantly, elevated levels of the phytohormone abscisic acid (ABA) were observed. Molecular and genetic studies revealed that ABA negatively influences defence to B05.10 and effects jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) levels. Susceptibility/resistance was determined by the antagonistic effect of ABA on JA, and this crosstalk required suppressing WRKY33 functions at early infection stages. This indicates that B. cinerea B05.10 promotes disease by suppressing WRKY33-mediated host defences.

Transcriptional events defining plant immune responses.

Rapid and massive transcriptional reprogramming upon pathogen recognition is the decisive step in plant-phytopathogen interactions. Plant transcription factors (TFs) are key players in this process but they require a suite of other context-specific co-regulators to establish sensory transcription regulatory networks to bring about host immunity. Molecular, genetic and biochemical studies, particularly in the model plants Arabidopsis and rice, are continuously uncovering new components of the transcriptional machinery that can selectively impact host resistance toward a diverse range of pathogens. Moreover, detailed studies on key immune regulators, such as WRKY TFs and NPR1, are beginning to reveal the underlying mechanisms by which defense hormones influence the function of these factors. Here we provide a short update on such recent developments.

Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100.

The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity.

Effect of Mild Cold Exposure on Cognition in Persons with Tetraplegia.

UNLABELLED: Persons with a cervical spinal cord injury (SCI) have impaired thermoregulatory mechanisms secondary to interrupted of motor, sensory, and sympathetic pathways. In this study, our primary aim was to determine the effect of cool temperature exposure on core body temperature (Tcore) and cognitive performance in persons with tetraplegia. Seven men with chronic tetraplegia (C3-C7, American Spinal Injury Association Impairment Scale [AIS] A-C) and seven able-bodied controls were exposed to 27°C temperature at baseline (BL) before being exposed to 18°C for ≤120 min (Cool Challenge). Rectal temperature (Tcore), distal skin temperatures (Tskavg), microvascular skin perfusion (LDFavg), and systolic blood pressure (SBP) were measured. Cognitive performance was assessed using Delayed Recall, Stroop Interference tests at the end of BL and Cool Challenge. After Cool Challenge, Tcore decreased -1.2±0.12°C (p<0.0001) in tetraplegics after an average of 109±15.9 min with no change in controls after 120 min. Tskavg declined in both groups, but decline was less in tetraplegics than in controls (-8.6±5.8% vs. -31.6±7.9%, respectively; p<0.0001). LDFavg declined only in controls (-72±17.9%; p<0.001). Plasma norepinephrine levels differed after Cool Challenge (tetraplegics vs. CONTROLS: 86±62 pg/mL vs. 832±431 pg/mL, respectively; p<0.01). SBP increased from BL to Cool Challenge only in controls (123±16 mm Hg to 149±17 mm Hg, respectively; p<0.01). Delayed Recall and Stroop Interference scores both declined in tetraplegics (-55±47.4%; p<0.05 and -3.9±3.8%; p<0.05, respectively), but not in controls. We conclude that persons with tetraplegia lack adequate thermoregulatory mechanisms to prevent downward drift in Tcore on exposure to cool temperatures. This decline in Tcore was associated with deterioration of working memory and executive function.

Differential expression pattern of an acidic 9/13-lipoxygenase in flower opening and senescence and in leaf response to phloem feeders in the tea plant.

BACKGROUND: Lipoxygenase (LOXs) is a large family of plant enzymes that catalyse the hydroperoxidation of free polyunsaturated fatty acids into diverse biologically active compounds, collectively named phyto-oxylipins. Although multiple isoforms of LOXs have been identified in a wide range of annual herbaceous plants, the genes encoding these enzymes in perennial woody plants have not received as much attention. In Camellia sinensis (L.) O. Kuntze, no LOX gene of any type has been isolated, and its possible role in tea plant development, senescence, and defence reaction remains unknown. The present study describes the isolation, characterization, and expression of the first tea plant LOX isoform, namely CsLOX1, and seeks to clarify the pattern of its expression in the plant's defence response as well as in flower opening and senescence. RESULTS: Based on amino acid sequence similarity to plant LOXs, a LOX was identified in tea plant and named CsLOX1, which encodes a polypeptide comprising 861 amino acids and has a molecular mass of 97.8 kDa. Heterologous expression in yeast analysis showed that CsLOX1 protein conferred a dual positional specificity since it released both C-9 and C-13 oxidized products in equal proportion and hence was named 9/13-CsLOX1. The purified recombinant CsLOX1 protein exhibited optimum catalytic activity at pH 3.6 and 25°C. Real-time quantitative PCR analysis showed that CsLOX1 transcripts were detected predominantly in flowers, up-regulated during petal senescence, and down-regulated during flower bud opening. In leaves, the gene was up-regulated following injury or when treated with methyl jasmonate (MeJA), but salicylic acid (SA) did not induce such response. The gene was also rapidly and highly induced following feeding by the tea green leafhopper Empoasca vitis, whereas feeding by the tea aphid Toxoptera aurantii resulted in a pattern of alternating induction and suppression. CONCLUSIONS: Analysis of the isolation and expression of the LOX gene in tea plant indicates that the acidic CsLOX1 together with its primary and end products plays an important role in regulating cell death related to flower senescence and the JA-related defensive reaction of the plant to phloem-feeders.

Pathophysiological preconditions promoting mixed "black" pigment plus cholesterol gallstones in a DeltaF508 mouse model of cystic fibrosis.

Gallstones are frequent in patients with cystic fibrosis (CF). These stones are generally "black" pigment (i.e., Ca bilirubinate) with an appreciable cholesterol admixture. The pathophysiology and molecular mechanisms for this "mixed" gallstone in CF are unknown. Here we investigate in a CF mouse model with no overt liver or gallbladder disease whether pathophysiological changes in the physical chemistry of gallbladder bile might predict the occurrence of "mixed" cholelithiasis. Employing a DeltaF508 mouse model with documented increased fecal bile acid loss and induced enterohepatic cycling of bilirubin (Am J Physiol Gastrointest Liver Physiol 294: G1411-G1420, 2008), we assessed gallbladder bile chemistry, morphology, and microscopy in CF and wild-type mice, with focus on the concentrations and compositions of the common biliary lipids, bilirubins, Ca(2+), and pH. Our results demonstrate that gallbladder bile of CF mice contains significantly higher levels of all bilirubin conjugates and unconjugated bilirubin with lower gallbladder bile pH values. Significant elevations in Ca bilirubinate ion products in bile of CF mice increase the likelihood of supersaturating bile and forming black pigment gallstones. The risk of potential pigment cholelithogenesis is coupled with higher cholesterol saturations and bile salt hydrophobicity indexes, consistent with a proclivity to cholesterol phase separation during pigment gallstone formation. This is an initial step toward unraveling the molecular basis of CF gallstone disease and constitutes a framework for investigating animal models of CF with more severe biliary disease, as well as the human disease.

Purification and characterization of a thermostable MnSOD from the thermophilic fungus Chaetomium thermophilum.

A thermostable superoxide dismutase (SOD) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum strain CT2 was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-sepharose, phenyl-sepharose hydrophobic interaction chromatography. The pure SOD had a specific activity of 115.77 U/mg of protein and was purified 7.49-fold, with a yield of 14.4%. The molecular mass of a single band of the enzyme was estimated to be 23.5 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 94.4 kDa, indicating that this enzyme was composed of four identical subunits of 23.5 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN and H2O2. Atomic absorption spectrophotometric analysis showed that the content of Mn was 2.05 microg/mg of protein and Fe was not detected in the purified enzyme. These results suggested that the SOD in C. thermophilum was the manganese superoxide dismutase type. N-terminal amino acid sequencing (10 residues) was KX (X is uncertain) TLPDLKYD. The N-terminal amino acid sequencing homologies to other MnSod also indicated that it was a manganese-containing superoxide dismutase. The SOD exhibited maximal activity at pH 7.5 and optimum temperature at 60 C. It was thermostable at 50 and 60 C and retained 60% activity after 60 min at 70 C. The half-life of the SOD at 80 C was approximately 25 min and even retained 20% activity after 30 min at 90 C.

Purification and characterization of two thermostable proteases from the thermophilic fungus Chaetomium thermophilum.

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and PhenylSepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65 degrees C. The PRO33 had a K(m) value of 6.6 mM and a V(max) value of 10.31 micromol/l/min, and PRO63 17.6 mM and 9.08 micromol/l/min, with casein as substrate. They were thermostable at 60 degrees C. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at 70 degrees C for 1 h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70 degrees C after 60 min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.

Purification, characterization, and molecular cloning of a thermostable superoxide dismutase from Thermoascus aurantiacus.

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS-PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.

[Cloning and expressing of cellulase gene (cbh2) from thermophilic fungi Chaetomium thermophilum CT2].

Chaetomium thermophilum CT2 can produce extracellular cellulase with industrial value. We designed two degenerate primers to amplify catalytic domain sequence of cellobiohydrolase II ( CBH II). Full length of cDNA was obtained by rapid amplification of cDNA ends technologies. DNA sequencing revealed that cbh2 has an open reading frame of 1428bp, which encodes a putative polypeptide of 476 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 53 kD and the cbh2 consists of a fungal-type carbohydrate binding domain (CBD) separated from a catalytic domain by a linker region rich in proline/serine/threonine. PCR product consisting of the entire CBH II coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. Highly efficient production of the cellobiohydrolase II was achieved in P. pastoris under the control of the AOX1 promoter, and the expressing level was 1.2 mg/mL by small-scale culturing. The recombinant cellobiohydrolase II was purified by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography. A molecular mass of the purified enzyme is 67 kD determined by SDS-PAGE and this is similar to the native cellobiohydrolase II purified from C. thermophilum CT2. The recombinant enzyme exhibited optimum catalytic activity at pH 4.0 and 50 degrees C respectively. It was thermostable at 50 degrees C and retained 50% of its original activity after 30 min at 70 d degrees C . The high level of fully active recombinant cellobiohydrolase II got from P. pastoris makes this expression system attractive for fermentor and industrial applications.