Characterization of Cloned Endoxylanase from Cellulomonas sp. NCIM 2353 Expressed in Escherichia coli

A 22-kDa xylanase encoded by a cloned gene (XCs16) of Cellulomonas was purified to homogeneity with an overall yield of 44%. It is a basic protein with a pI of 8.1 and has a K _m and V _max of 3 mg/ml and 1150 μmoles/mg/min, respectively, for oat spelt xylan at 55°C and pH 5.8. Homologous xylanase from Cellulomonas could be identified with antibodies raised against purified xylanase encoded by XCs16. The enzyme from Cellulomonas also exhibited identical temperature and pH optimum and had a molecular weight of 23 kDa. Modification of tryptophan residue of purified xylanase resulted in the loss of xylanase activity. This loss could be reversed by the addition of substrate, indicating the involvement of tryptophan residue in the catalytic site. Received: 12 April 1996 / Accepted: 28 October 1996     

Cloning and expression of a β-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme

Abstract A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA , was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 °C) optima were determined.     

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli

An amylase gene was identified in a Streptococcus bovis 033 gtWESB genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.     

Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis

AIMS: The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus. METHODS AND RESULTS: The 750 bp xylanase gene was amplified and subcloned into the unique NheI restriction enzyme site of pMG36e and subsequently transformed into competent Escherichia coli XLI-blue MRF cells and Lactococcus lactis cells. Bacterial culture containing pMG36e-Xy has an enzyme activity of 390 microg xylose ml(-1) culture 30 min(-1), respectively, when compared with 40 microg xylose ml(-1) culture 30 min(-1) for the negative control (plasmidless strain). CONCLUSIONS: The thermostable xylanase gene was successfully expressed in both E. coli and L. lactis. The activity of xylanase can be easily detected by the formation of visible clearing zones around the transformed colonies on Remazol Brilliant Blue-Xylan (RBB-Xylan) agar media. However, there were some significant differences in the optimum growth temperature and plasmid stability in the new clones. SIGNIFICANCE AND IMPACT OF THE STUDY: The constructed reporter vector has the potential to be used as a reporter system for Lactococcus as well as E. coli, and it is an addition to the pool of lactococcal vector systems.     

Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM‐2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl₂ as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H₂ energy was examined with seven H₂‐producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H₂ production performance with the hydrolysate of SCB and xylan. The cumulative H₂ production and H₂ yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H₂/g reducing sugar (or 1.91 mmol H₂/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H₂ production and H₂ yield of 87.02 mL/L and 5.03 mmol H₂/g reducing sugar (or 4.01 mmol H₂/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Multiple mechanisms of uranium immobilization by Cellulomonas sp. strain ES6

Removal of hexavalent uranium (U(VI)) from aqueous solution was studied using a Gram‐positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non‐growth conditions in bicarbonate and PIPES buffers. Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate‐based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X‐ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non‐uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)‐phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U‐free controls suggesting simultaneous precipitation of U and PO. In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High‐resolution transmission electron microscopy (HR‐TEM) and energy dispersive X‐ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone‐2,6‐disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non‐uraninite U(IV) phase. Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6. Biotechnol. Bioeng. 2011;108: 264–276. © 2010 Wiley Periodicals, Inc.     

Trehalose, a cytoplasmic reserve disaccharide of Cellulomonas sp. DSM20108: Its identification, carbon-source-dependent accumulation, and degradation during starvation

Abstract During growth on complete medium, as well as on mineral salts medium with different carbon sources, Cellulomonas sp. synthesized trehalose (α- d -glucopyranosyl-α- d -glucopyranoside). This non-reducing disaccharide was accumulated intracellularly. Its specific concentration (up to 3 μmol per mg crude extract protein) reached a maximum during cultivation and was shown to be dependent on the carbon source used. During starvation at 30°C, trehalose was degraded in vivo at an average initial specific rate of about 0.5 μmol per mg crude extract protein per day. To our knowledge this is the first time that trehalose has been reported to be an energy storage compound in a prokaryotic microorganism.     

Degradation of microcrystalline cellulose: synergism between different endoglucanases of Cellulomonas sp. ATCC 21399

Three immunologically and enzymatically distinct endoglucanases of Cellulomonas sp. ATCC 21399 were purified previously. Endoglucanase A and endoglucanase B acted synergistically on microcrystalline cellulose (Avicel), whereas no synergistic action was observed between endoglucanase B or endoglucanase C. Only endoglucanase A was capable of hydrolyzing Avicel when acting alone and this enzyme resulted in "short fiber formation" when acting on Avicel. The end product of hydrolysis of acid swollen Avicel produced by the three endoglucanases was in all cases dominated by cellobiose and showed lower content of glucose and cellotriose. Higher cellodextrins appeared as transient end products. The results indicate that the function of endoglucanase A in the cellulase system of Cellulomonas might be very similar to the function of the cellobiohydrolases of Trichoderma reesei.     

Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli.

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.     

猴头菌UDP-葡萄糖-4-差向异构酶基因克隆表达及酶学性质

通过实验室前期对诱变菌株猴头菌321的多组学分析结果,获得了一个多糖合成过程中起关键作用的UDP-葡萄糖-4-差向异构酶基因（UDP-glucose-4-epimerase,UGE）,并在大肠杆菌E. coli BL21（DE3）中进行了异源表达。通过筛选最优的目的蛋白诱导表达条件后,通过镍柱亲和层析纯化后获得高纯度目的蛋白,并对目的蛋白进行了酶学性质的研究,明确了其生物学性质和动力学参数,为其开发利用提供理论参考。     

Separation and characterization of endoglucanases from culture filtrates of Cellulomonas sp.

Summary Six major components exhibiting endo-1,4-\-d-glucanase activity were partially purified from culture filtrates of a newly isolated Cellulomonas sp. using ion-exchange chromatography. Molecular weights (44,000 to 140,000), pH optima (6.0 to 7.0), temperature optima (40 to 50°C), half-life, energy of activation, K _mand other kinetic parameter investigations indicate the existence of 6 different endoglucanases.Further support for this assumption comes from inhibition studies, whereby glucose inhibited the enzyme activities between 15 and 50% at a concentration of 0.034% (1.65 mM) and cellobiose between 0 and 50% at a concentration of 0.1% (2.92 mM). Of all the metals (Hg²⁺, Co²⁺, Cu²⁺, Ca²⁺, Mg²⁺, Zn²⁺, Fe³⁺) tested, only Hg²⁺ exhibited a 55% inhibition at 5.0 mM.     

Purification and partial characterization of three extracellular cellulases from Cellulomonas sp.

Three extracellular cellulases have been purified from cultures of Cellulomonas. One was found in solution in the cell-free supernatant and two others were found to be bound to the cellulose added as a carbon source. The free enzyme and one of the cellulose-bound enzymes bind to Sephadex. The two cellulose-bound enzymes are glycosylated. The three enzymes behave as endocellulases towards soluble carboxymethyl-cellulose and have little activity on cellulose powder.     

来源于耐碱真菌Pseudallescheria sp. JSM-2的碱性木聚糖酶基因的克隆、表达及其性质研究

从造纸废水中分离得到的耐碱真菌Pseudallescheria sp. JSM-2的DNA为模板,利用同源克隆和TAIL-PCR的方法,获得了一个碱性木聚糖酶基因xyl11-1。该基因DNA和cDNA分别为797 bp和678 bp。该基因的推测蛋白N-端有一个18个氨基酸的信号肽序列和一个含207个氨基酸的成熟蛋白。编码成熟蛋白的cDNA序列在毕赤酵母GS115中重组表达后,进一步纯化并进行酶学性质测定。重组XYL11-1的最适pH为6.5,在pH 4.5~9.0范围有50%以上的酶活;在pH 4.5~12.0范围具有良好的pH稳定性;最适温度为50℃;以燕麦木聚糖为底物,比活为2 618 U/mg;且对中性和碱性蛋白酶具有极好的抗性。该酶作用底物范围广,包括各种木聚糖、纤维素和葡聚糖,易于工业化发酵生产,具有在纸浆脱墨、动物饲料、鱼类饵料中的应用潜力。     

Comparative characterization of two serine endopeptidases from Nocardiopsis sp. NCIM 5124

A protease-producing, crude oil degrading marine isolate was identified as Nocardiopsis sp. on the basis of the morphology, cell wall composition, mycolic acid analysis and DNA base composition. The Nocardiopsis produces two extracellular proteases, both of which are alkaline serine endopeptidases. Protease I was purified to homogeneity by chromatography on CM-Sephadex at pH 5.0 and pH 9.0. Protease II was purified using DEAE-cellulose, Sephadex G-50, phenyl-Sepharose and hydroxyapatite chromatography. Protease I and II had almost similar M(r) of 21 kDa (Protease I) and 23 kDa (Protease II), pI of 8.3 and 7.0 respectively with pH and temperature optima for activity between 10.0 and 11.0 and about 60 degrees C. Specific activities were 152 and 14 U/mg respectively on casein. However, Protease I was antigenically unrelated to Protease II. Both proteases were endopeptidases and required extended substrate binding for catalysis. Both proteases had collagenolytic and fibrinolytic activity but only Protease I had elastinolytic activity. The proteases were chymotrypsin-like with respect to their amino acid compositions and N-terminal sequences.     

Fermentative production of extracellular amylase from novel amylase producer,Tuber maculatum mycelium,and its characterization

Abstract

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22?±?2?°C) under static conditions which resulted in 1.41?U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50?°C. The enzyme showed good thermostability at 50?°C by retaining 98% of the maximal activity after 100?min of incubation. The amylase activity was marginally enhanced in presence of Cu²⁺ and Na⁺ and slightly reduced by K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Co²⁺, Zn²⁺, and Mn²⁺ ions at 1?mM concentration.     

一株假单胞菌(Pseudomonas sp.)L-1菌株γ-丁基甜菜碱羟化酶基因bbh 的克隆、表达及酶学性质

【目的】γ-丁基甜菜碱羟化酶是生物体内合成L-肉碱的关键酶。从假单胞菌(Pseudomonas sp.)L-1中克隆γ-丁基甜菜碱羟化酶基因,实现其在大肠杆菌(Escherichia coli)中的高效表达,并对表达产物进行酶学性质分析,为生物转化生产L-肉碱奠定基础。【方法】通过PCR克隆γ-丁基甜菜碱羟化酶基因,并将其开放阅读框(ORF)克隆至融合表达载体pET-15b;表达产物经His.Bind Resin纯化后对BBH进行酶学性质及三维空间结构分析;并以静止细胞进行L-肉碱的转化。【结果】成功地克隆了一个γ-丁基甜菜碱羟化酶基因bbh(GenBank:JQ250036),并实现了其在E.coli中的高效表达。融合蛋白以同源二聚体的形式存在,单个亚基的分子量约46.5 kDa,最适反应温度为30℃,最适反应pH为7.5。该酶在45℃以下稳定。在pH6.0时该酶有最高的pH稳定性。以表达bbh基因的重组大肠杆菌静止细胞转化L-肉碱,L-肉碱产量可达12.7mmol/L。【结论】Pseudomonas sp.L-1γ-丁基甜菜碱羟化酶与现有报道的bbh基因有较大的差异。由该基因表达的γ-丁基甜菜碱羟化酶能有效地转化γ-丁基甜菜碱生成L-肉碱。本研究不仅丰富了γ-丁基甜菜碱羟化酶基因资源,而且为L-肉碱的生物转化提供了一种新的转化方案。     

Identification and characterization of a novel spermidine/spermine acetyltransferase encoded by gene ste26 from Streptomyces sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which can bind IL-1R specifically and exhibits anti-rheumatic arthritis activity in vivo. With the Ebosin biosynthesis gene cluster (ste) consisting of 27 ORFs identified previously the focus of this study was to characterize the protein encoded by ste26 gene. After cloning and expressing ste26 in Escherichia coli BL21, we purified the recombinant Ste26 protein and revealed its ability of transferring the acetyl group from AcCoA to spermidine and spermine, with spermine being the preferred substrate. Therefore Ste26 has been determined to be a spermidine/spermine acetyltransferase which can use spermine (Km of 72.1 ± 7.4 μM), spermidine (Km of 147.2 ± 11 μM), AcCoA (Km of 45.7 ± 2.5 μM) and poly-l-lysine (Km of 99.7 ± 11 μM) as substrates. The optimum pH, temperature and time for the activity have been shown to be 7.5, 37°C and 10 min, respectively. This is the first spermidine/spermine acetyltransferase characterized in Streptomyces and its function in Ebosin biosynthesis is discussed.     

Molecular cloning and expression of a xylanase gene from Cellulomonas sp. into Escherichia coli

Summary The xylanase gene of Cellulomonas sp. NCIM 2353 was cloned in pUC 18 and selected by growth on xylan as the sole carbon source. The functional clone harboured the recombinant plasmid with an insert of 1.42 kbp, as determined by restriction mapping and Southern hydridization. The clone secreted a xylanase of 45 000 mol. wt. as determined by Western blot analysis using specific antixylanase antibodies. The DNA insert carried the full structural gene along with its promoter and possibly regulatory sequences, since xylanase activity in the clone Cs11 was inducible by xylan. Offprint requests to: D. N. Deobagkar