Cloning and expression of a novel thermostable cellulase from newly isolated <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain I15

A novel bacterial strain with high cellulase activity (2.82 U/ml) was isolated, and then identified by its morphological character and 16S rRNA sequence, and named Bacillus subtilis strain I15. The extracellular thermostable cellulase exhibited the maximum activity at 60°C and pH 6.0. It was very stable since more than 90% of original CMCase activity was maintained at 65°C after incubation for 2 h. The cellulase gene, celI15, was cloned and extracellularly expressed by Escherichia coli BL21 (DE3), which encoded the extracellular protein of about 52 kDa. The extracellular activity of CelI15 from E. coli BL21 was up to about 6.78 U/ml, and all the other properties were almost the same as that from the wild-type strain.     

Gene Cloning of Endoglucanase Cel5A from Cellulose-Degrading <Emphasis Type="Italic">Paenibacillus xylanilyticus</Emphasis> KJ-03 and Purification and Characterization of the Recombinant Enzyme

The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.     

The endoglucanase from <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BEC-1 bears halo-tolerant,acidophilic and dithiothreitol-stimulated enzyme activity

A Bacillus subtilis strain BEC-1 demonstrating high carboxymethylcellulose-degrading activity was isolated from the forest soil sample. In order to characterize the biochemical specialty of its cellulase, the endoglucanase gene egl173 was cloned from this strain and was expressed in Escherichia coli. The gene encoded a protein of 499 amino acids with a molecular weight of 64 kDa. The purified Egl173 could hydrolyze both soluble and insoluble celluloses with distinct activities. This enzyme showed the highest enzyme activity at pH 4, maintained at least 85% activity in the pH range of 3–7, displayed maximum activity at 60°C and was highly stable between 30 and 60°C. It was found that this endoglucanase was increasedly active and retained its high stability after incubation with 5 M NaCl or 3 M KCl for 24 h. Furthermore, after incubation with 10 mM of dithiothreitol, the enzyme activity was significantly enhanced (125% of the control level). In the presence of diverse metal ions (except mercury and manganese cations), organic solvents, surfactants (except SDS) and chelating agent, this enzyme kept more than 85% active. This halo-tolerant, acidophilic and highly stable endoglucanase is prospectively to be exploited as the advanced enzymatic product for diverse industrial applications.     

Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Gene Cloning and Characterization of a Thermostable Phytase from Bacillus subtilis US417 and Assessment of its Potential as a Feed Additive in Comparison with a Commercial Enzyme

An extracellular phytase from Bacillus subtilis US417 (PHY US417) was purified and characterized. The purified enzyme of 41 kDa was calcium-dependent and optimally active at pH 7.5 and 55°C. The thermal stability of PHY US417 was drastically improved by calcium. Indeed, it recovered 77% of its original activity after denaturation for 10 min at 75°C in the presence of 5 mM CaCl₂, while it retained only 22% of activity when incubated for 10 min at 60°C without calcium. In addition, PHY US417 was found to be highly specific for phytate and exhibited pH stability similar to Phyzyme, a commercial phytase with optimal activity at pH 5.5 and 60°C. The phytase gene was cloned by PCR from Bacillus subtilis US417. Sequence analysis of the encoded polypeptide revealed one residue difference from PhyC of Bacillus subtilis VTTE-68013 (substitution of arginine in position 257 by proline in PHY US417) which was reported to exhibit lower thermostability especially in the absence of calcium. With its neutral pH optimum as well as its great pH and thermal stability, the PHY US417 enzyme presumed to be predominantly active in the intestine has a high potential for use as feed additive.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

High-level expression,purification and characterization of a recombinant medium-temperature <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase (AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml⁻¹) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg⁻¹ having optimal activity at pH 6.0 and 60°C.     

Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.     

Construction of the bifunctional enzyme cellulase-β-glucosidase from the hyperthermophilic bacterium <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

An artificial bifunctional enzyme, cellulase-β-glucosidase, was prepared by gene fusion from the hyperthermophilic bacterium Thermotoga maritima MSB8. The fusion protein exhibited both cellulase (Cel5C) and β-glucosidase (BglB) activity when the bglB gene was fused to downstream of cel5C, but not when cel5C was fused to downstream of bglB. The specific activity of the bifunctional enzyme was 70% lower than that of cellulase or β-glucosidase. The fusion enzyme was purified, and the MW was estimated as 114 kDa. The fusion enzyme displayed optimum cellulase activity at pH 8.0 and 70°C over 30 min, and optimal β-glucosidase activity at pH 7.0 and 80°C over 30 min.     

Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> LD-8547

A fibrinolytic enzyme producing strain Bacillus subtilis LD-8 was isolated from douchi, a traditional Chinese soybean-fermented food. After mutagenesis treatments by UV, NTG (N-methyl-N′-nitroso-N-nitroso-guanidine) and γ-radiation, a high fibrinolytic enzyme producing strain B. subtilis LD-8547 was obtained. Under optimum condition, LD-8547 was able to yield the average fibrinolytic activity of 4220 U/mL in 15 L fermenter. The strong fibrin-specific enzyme was purified from supernatant of B. subtilis LD-8547 culture broth using the combination of various steps. The optimal temperature and pH value of this fibrinolytic enzyme were 50 °C and 8.0, respectively. The molecular weight was about 30 kDa measured by SDS-PAGE. The amidolytic activity of this fibrinolytic enzyme was inhibited completely by 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), but EDTA and EGTA did not affect the enzyme activity. The apparent K _m and V _max values were 0.521 mmol/L and 0.049 mmol/min, respectively. In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent.     

Purification and characterization of an extracellular keratinase from a hornmeal-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MTCC (9102)

Native proteolytic microorganisms were isolated from the hornmeal, which is a product obtained by treatment of horns and hoofs with steam under high pressure. Keratinolytic activities of these organisms were screened in mineral salt medium with 1% hornmeal. Bacillus subtilis MTCC (9102), a keratinase-producing organism causing extensive degradation of hornmeal has been identified. Keratinase was purified (45-fold) by ion exchange, and gel filtration chromatography. Among the keratinases produced by the various organisms, keratinase from the Bacillus subtilis strain reported by us was found to have a molecular weight range between 64 and 69 kDa and high activity in the pH range between 5 and 7, with maximum activity at pH 6.0 and at an optimum temperature of 40°C. It remained stable up to 70°C. The keratinase activity was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and 1 10-phenanthroline, and remained unaffected by phenylmethanesulfonyl fluoride (PMSF, relative activity: 93%), whereas iodoacetamide inhibited considerably. Zinc, magnesium, calcium, manganese, and nickel were found to enhance the enzyme activity, whereas mercury and copper inhibited its activity completely. The keratinolytic metalloprotease from native Bacillus subtilis differed from the other serine proteases. It may have potential applications in the bioconversion of keratinous wastes and eco-friendly dehairing in the leather industry.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45 kDa, 55 °C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138 U mL^?1 at 44 h, and the productivity reached 48.58 U (mL h)^?1 through the homologous overexpression of strain B-pN-pelA in a 7.5 L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50 °C for 4 h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

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.

     

Screening and Identification of New Isolate: Thermostable <Emphasis Type="Italic">Escherichia coli</Emphasis> with Novel Thermoalkalotolerant Cellulases

A cellulase-producing bacterium strain was isolated from soil that produced novel thermoalkalotolerant cellulases after growth on CMC-Na agar screening plate at 37°C. It was identified as Escherichia coli using the method of 16S rRNA and intergenic spacer gene analysis combined with morphological, physiological, and biochemical tests. Three major components of the cellulases [carboxymethyl cellulase (CMCase), filter paper cellulase, and β-glucosidase] were produced with maximal activities (0.23, 0.08, and 0.15 U/ml) and maximum specific activities 4.13, 0.56, and 0.50 U/mg protein after 72, 96, and 120 h growth, respectively. Maximum CMCase activity was measured at 50°C and pH 6.0, respectively, and it also retained more than 60% of its maximal activity for at least 20 min at 50–70°C and 10 min at 80°C, respectively, and retained approximately 50% of its maximal activity after incubating at 90°C for 10 min. The enzyme could be applied in bioconversion of lignocellulosic agricultural wastes.     

Optimization of laboratory cultivation conditions for the synthesis of antifungal metabolites by bacillus subtilis strains

In order to achieve the optimal number of colony forming units and a high level of antifungal metabolites synthesis, we carried out the periodic cultivation of the Bacillus subtilis BZR 336 g and Bacillus subtilis BZR 517 strains at various pH and temperature levels. In the experiment for determining the optimal temperature, the maximum titer of B. subtilis BZR 336 g bacterium (1.6–1.7 × 10⁹ CFU/ml) was recorded at a cultivation temperature of 20–25 °C. For B. subtilis BZR 517 strain, the temperature turned out to be optimal at 30 °C: the titer was 8.9 × 10⁸ CFU/ml. The maximum antifungal activity of B. subtilis BZR 336 g strain against the test culture of Fusarium oxysporum var. orthoceras was observed at a cultivation temperature of 20–25 °C; for B. subtilis BZR 517 strain, 25–30 °C. When determining the optimal pH level, it was found that a high titer of B. subtilis BZR 336 g strain cells was determined at pH 8.0 (2.7 × 10⁹ CFU/ml), for B. subtilis BZR 517 strain it was at pH 6.0–8.0 (1.0 × 10⁹ CFU/ml). The maximum antifungal activity was noted with the same indicators. Chromatographic and bioautographic analyses suggest that the synthesized antifungal metabolites belong to surfactin and iturin A. The data obtained in this research can be used in the development of the technology for the production of effective biofungicides to protect crops against Fusarium pathogens.     

A bifunctional cellulase–xylanase of a new Chryseobacterium strain isolated from the dung of a straw‐fed cattle

A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase–xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 μmol min^?1 mg^?1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 μmol min^?1 mg^?1 at pH 8, 90 °C. The activity level and thermophilicity are in the front rank of all the known cellulases and xylanases. Core hydrophobicity had a positive effect on the thermophilicity of this enzyme. When similar quantity of enzymatic activity units was applied on the straws of wheat, rice, corn and oilseed rape, CbGH5 could obtain 3.5–5.0× glucose and 1.2–1.8× xylose than a mixed commercial cellulase plus xylanase of Novozymes. When applied on spent mushroom substrates made from the four straws, CbGH5 could obtain 9.2–15.7× glucose and 3.5–4.3× xylose than the mixed Novozymes cellulase+xylanase. The results suggest that CbGH5 could be a promising candidate for industrial lignocellulosic biomass conversion.