Properties of a genetically reconstructed Prevotella ruminicola endoglucanase.

A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonospora fusca E2 cellulose-binding domain at its C terminus joined to the Prevotella ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not.     

The cellular location of Prevotella ruminicola beta-1,4-D-endoglucanase and its occurrence in other strains of ruminal bacteria.

Prevotella ruminicola B(1)4, TC1-1, TF1-3, and TS1-5 all produced immunologically cross-reacting 88- and 82-kDa carboxymethyl cellulases (CMCases). P. ruminicola 23, 118B, 20-63, and 20-78 had much lower CMCase activities, and Western blots (immunoblots) showed no cross-reaction with the B(1)4 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced CMCase, but these enzymes were smaller and did not cross-react with the B(1)4 CMCase antiserum. The B(1)4 CMCase antiserum inhibited the B(1)4, TC1-1, TF1-3, and TS1-5 CMCase activities and agglutinated these cells, but it had no effect on the other strains or species. On the basis of these results, the B(1)4 CMCase is a strain-specific enzyme that is located on the outside surface of the cells. P. ruminicola B(1)4 cultures, grown on sucrose, did not have significant CMCase activity, but these cells could bind purified 88- and 82-kDa CMCase but not 40.5-kDa CMCase. Because the 40.5-kDa CMCase is a fully active, truncated form of the CMCase, it appears that the N-terminal domain of the 88-kDa B(1)4 CMCase anchors the CMCase to the cells. Cells grown on cellobiose produced at least 10-fold more CMCase than the sucrose-grown cells, and the cellobiose-grown cells could only bind 15% as much CMCase as sucrose-grown cells. Virtually all of the CMCase activity of exponentially growing cultures was cell associated, but CMCase activity was eventually detected in the culture supernatant. On the basis of the observation that the 88-kDa CMCase was gradually converted to the 82-kDa CMCase when cultures reached the stationary phase without a change in specific activity, it appears that the 82-kDa protein is probably a proteolytic degradation product of the 88-kDa CMCase.     

产高温纤维素酶木霉菌株的筛选、鉴定及酶学性质研究

从稻草堆肥中筛选得到一株产高温纤维素酶的霉菌M1,通过形态学观察和分子生物学鉴定,确定其为木霉属(Trichoderma)。在稻草液体发酵培养基中,木霉M1的CMC酶(carboxymethyl cellulase,CMCase)合成模式为同步合成型。酶学性质研究表明,此CMC酶的最适反应pH为4.4,在pH 4.0～6.0保温4h仍可保持95%以上的酶活力;其最适反应温度为75℃,在50℃下保温4h,可保持87%的酶活力;60℃下保温4h,可保持65%的酶活力,具有较好的热稳定性。     

麝鼠肠道纤维素分解菌的分离鉴定及其酶学特性分析

本研究旨在从麝鼠(Ondatra zibethicus)肠道中分离出高效分解纤维素的菌株,为开发纤维素分解菌微生物制剂提供菌种资源。本研究利用以羧甲基纤维素钠(CMC-Na)为单一碳源的培养基,从麝鼠盲肠内分离出--株高效分解纤维素的菌株WJ-3,并对该菌株进行形态鉴定、生理生化鉴定和16S.rDNA分子鉴定。对菌株WJ-3所产羧甲基纤维素酶(CMCase)进行酶学特性实验,分析此纤维素酶的最佳反应pH和最佳反应温度,以及此纤维素酶对不同温度和不同酸碱度的耐受性。结果表明,菌株WJ-3属于空气芽孢杆菌(Bacillus aerius),并将其命名为Bacillus aerius WJ-3。菌株WJ-3所产羧甲基纤维素酶在pH 4.0~6.0的范围内反应时,酶活性随pH值升高而增加,其最佳反应pH为6.0,且此纤维素酶在pH4.0~8.0范围内保存30min后均能保持80%以上的相对酶活性:菌株WJ-3所产羧甲基纤维素酶在温度30~50 ℃范围内反应时,随温度上升酶活性逐渐增加,在50 ℃时酶活性最高,之后随温度的升高酶活性逐渐下降,且纤维素酶在此温度范围内保存30 min后均能保持较高的酶活性。综上所述,菌株Bacillus aerius WJ-3所产羧甲基纤维素酶的酶活性较高,并且此纤维素酶的耐酸碱性及热稳定性良好,是具有一定利用价值的菌种资源。     

Studies on carboxymethyl cellulase produced by an alkalothermophilic actinomycete

A novel alkalothermophilic actinomycete having optimum growth at pH 9 and 50 degrees C was isolated from self-heating compost from the Barabanki district of Uttar Pradesh, India. Based on its morphology, susceptibility of spores to heat and novobiocin, guaninecytosine content of chromosomal DNA and cell wall composition, the organism was classified under Thermomonospora. The alkalothermophilic actinomycete produced 23 IU/ml carboxymethyl cellulase (CMCase). The CMCase was purified by fractional ammonium sulphate precipitation followed by cellulose affinity chromatography and Sephacryl S-200 gel filtration. The CMCase had a molecular weight of 38 KD and pI of 4.1. The enzyme exhibited optimum activity at pH 5 and temperature 50 degrees C. The CMCase showed pH stability in the range 7-10. The enzyme retained 100% activity at 50 degrees C for 72 h and had half-lives of 7 and 3 h at 60 degrees C and 70 degrees C, respectively. The CMCase was stable in the presence of commercial detergents such as Ariel, Henko and Surf Excel, indicating its potential as an additive to laundry detergents.     

白蚁肠道内纤维素酶产生菌的筛选及其酶活性

从黑翅土白蚁(Odontotermes formosanus Shiraki)肠道内筛选具有纤维素降解能力的细菌,并研究其酶活性。结果表明:筛选得到5株菌株,活力较高的菌株CMC-4被鉴定为土白蚁特拉布尔希氏菌Z-4(Trabulsiella odontotermitis ZJSRU-4)。同时对菌株T.odontotermitis ZJSRU-4进行了系统的研究,它具有完整的纤维素酶系统,主要产羧甲基纤维素酶(CMCase)和β-葡萄糖苷酶,滤纸酶的活力较低。在以羧甲基纤维素钠为碳源的培养基中培养36 h,发酵液中CMCase的比酶活达到20.8 U/m L,培养44 h,β-糖苷酶的比酶活达到18.2 U/m L。CMCase和β-葡萄糖苷酶作用的p H分别为6.0和6.5,它们作用的最适温度都为40℃。该菌对纤维质原料具有降解能力,具有潜在的应用价值。     

An analysis of the extracellular xylanases and cellulases of Butyrivibrio fibrisolvens H17c

The extracellular xylanase and cellulase components of Butyrivibrio fibrisolvens H17c were investigated. Two major peaks of enzyme activity were eluted by hydroxylapatite chromatography and designated complex A (CA), having cellulase activity, and complex B (CB) having predominantly xylanase activity but with some activity on carboxymethyl cellulose (CMC). CB was further purified on a DE-52 column and subjected to gel filtration. The xylanase and CMCase activities eluted in a single peak with an apparent molecular mass greater than thyroglobulin (Mr 669,000). CMC xymograms of polyacrylamide gels electrophoresed under non-denaturing conditions indicated the presence of five bands with CMCase activity from CA and eight from CB. Xylan xymograms under the same conditions indicated the presence of four bands of activity in CB. Under mild denaturing conditions the xylanase activity in CB was found in 11 bands with molecular mass ranging from 45 to 180 and the CMCase activity in three bands with molecular mass ranging from 45 kDa to 60 kDa. This indicates that CB exists as a multi-subunit protein aggregate of xylanases, some of which also have cellulase activity.     

Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.     

Identification of Mycelium-Associated Cellulase from Streptomyces reticuli

Among 180 Streptomyces strains tested, 25 were capable of hydrolyzing microcrystalline cellulose (Avicel) at 30°C. Streptomyces reticuli was selected for further studies because of its ability to grow at between 30 and 50°C on Avicel. Enzymatic activities degrading Avicel, carboxymethyl cellulose, and cellobiose were found both in the culture supernatant and in association with the mycelium and crystalline substrate. The bound enzymes were efficiently solubilized by repeated washes with buffer of low ionic strength (50 mM Tris hydrochloride [pH 7.5]) and further purified by fast protein liquid chromatography. A high-molecular-weight Avicelase of >300 kilodaltons could be separated from carboxymethyl cellulase (CMCase) and β-glucosidase activities (molecular mass, 40 to 50 kilodaltons) by gel filtration on Superose 12. The CMCase fraction was resolved by Mono Q anion-exchange chromatography into two enzymes designated CMCase 1 and CMCase 2. The β-glucosidase activity was found to copurify with CMCase 2. The purified cellulase components showed optimal activity at around pH 7.0 and temperatures of between 45 and 50°C. Avicelase (but not CMCase) activity was stimulated significantly by the addition of CaCl₂.     

Bacterial Cell Surface Display of an Enzyme Library for Selective Screening of Improved Cellulase Variants

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.     

Purification and characterization of cellulase fromBacillus thermoalcaliphilus isolated from a termite mound

An extracellular carboxymethyl cellulase (CMCase) was purified to homogeneity fromBacillus thermoalcaliphilus sp. nov. The cellulase was composed of a single subunit with molar mass of 46 kDa. The apparentK _m was at 3.5 mg cellulose per mL. Its optimum pH was 8.5, it was most stable at pH 8.5–9.5 but 50% of enzyme activity was present after 30 min at pH 11.0. The activity was highest at 70°C.     

纤维素酶中具有壳聚糖水解酶活性成分的鉴定

在壳聚糖酶的研究过程中,目前已发现37种酶具有非专一性地降解壳聚糖的能力[1].对这些非专一性酶水解壳聚糖的机理有两种看法:一些人认为,由于这些酶大都来自商业酶制剂,未经过进一步的纯化,故有人认为其中所含的少量杂质可能是产生水解活力的原因;但也有人认为,在所有的酶制剂中都存在同一种杂质似乎是不可能的,因为这些酶来源于广泛的微生物、真菌、哺乳动物和植物等.众所周知,酶具有高度的专一性,即对所催化的反应和底物有严格的选择性,一种酶往往只能催化一种或一类反应;有如此多的不同种类的酶能非专一性地水解壳聚糖.因而探讨具有水解…     

Expression and secretion of carboxymethyl cellulase in Bacillus subtilis by Lactobacillus casei lactate dehydrogenase gene promoter

The secretion ability of the signal peptide of carboxymethyl cellulase (CMCase) was combined with the high expression ability of the Lactobacillus casei lactate dehydrogenase gene (ldh) promoter sequence (P) in Bacillus subtilis RM125. The CMCase was successfully secreted to the culture medium and the enzyme activity by P (11.2 U/ml) was approximately 4 fold higher than by its own promoter (2.7 U/ml).     

Effect ofMyrothecium sp. cellulase on different types of cellulose and cellulosic materials

Three types of cellulase preparations were applied to different types of cellulose and cellulosic materials. The action of these types of cellulase on cellulose powder was increased with the increase of enzyme concentration. Both carboxymethyl cellulose (CMC) and sodium carboxymethyl cellulose (Na-CMC) released high amounts of reducing sugar as affected by cellulase application. Different types of paper pulp were moderately hydrolyzed, while agricultural wastes were slightly hydrolyzed. Vegetable and fruits cellulose were equally hydrolyzed but at low rate. Pretreatment of cellulose or cellulosic materials by grinding or by swelling with phosphoric acid gave rise to increased hydrolysis by the enzyme. Cellobiose was detected chromatographically as an intermediate product of hydrolysis of both cellulose and carboxymethyl cellulose with glucose.     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum.

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes

Avicelase, carboxymethyl cellulase (CMCase), and β-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.     

Purification and characterisation of alkaline cellulase produced by a novel isolate,<Emphasis Type="Italic"> Bacillus sphaericus</Emphasis> JS1

A novel strain of Bacillus sphaericus JS1 producing thermostable alkaline carboxymethyl cellulase (CMCase; endo-1,4--glucanase, E.C. 3.2.1.4) was isolated from soil using Horikoshi medium at pH 9.5. CMCase was purified 192-fold by (NH₄)₂SO₄ precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 23%. The CMCase is a multimeric protein with a molecular weight estimated by native-PAGE of 183 kDa. Using SDS-PAGE a single band is found at 42 kDa. This suggests presence of four homogeneous polypeptides, which would differentiate this enzyme from other known alkaline cellulases. The activity of the enzyme was significantly inhibited by bivalent cations (Fe³⁺ and Hg²⁺, 1.0 mM each) and activated by Co²⁺, K⁺ and Na⁺. The purified enzyme revealed the products of carboxymethyl cellulose (CMC) hydrolysis to be CM glucose, cellobiose and cellotriose. Thermostability, pH stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases make this enzyme potentially useful in laundry detergents.     

Resuscitation of viable but non‐culturable bacteria to enhance the cellulose‐degrading capability of bacterial community in composting

Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation‐promoting factors (Rpfs) due to entering a viable but non‐culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose‐degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl‐cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.     

芽孢杆菌074碱性纤维素酶的研究——Ⅰ.菌种的分离、筛选及发酵条件

从碱性土样中,分离到产碱性纤维素酶的细菌134株。经摇瓶初筛、复筛后,得到一株芽孢杆菌074,在pH9条件下能产生具有较高活性的纤维素酶。该菌株的最适生长pH为中性,最适生长温度为32℃。NaC1对酶的产生影响较大。羧甲基纤维素钠(CMC-Na)对酶的产生有一定促进作用,但不是唯一碳源。用最佳培养基和培养条件,48小时酶活性最高可达6u/ml。