Studies on the β-glucosidase and endo-1, 4-glucanase activities from Clostridium papyrosolvens

Activities of endo-β-4-glucanase and β-glucosidase from Clostridium papyrosolvens (CECT 747) were studied under different conditions. No indications were found for the presence of more than one β-glucosidase after ion exchange and ammonium sulphate fractionation. β-Glucosidase showed activity against p-nitrophenyl-β-D-glucopyranoside and cellobiose but was not inhibited by δ-gluconolactone. Independent cellobiose phosphorylase activity could also be detected. The β-glucosidase was essentially intracellular. By the use of stationary phase culture assays, enzyme induction at low concentrations of cellobiose was observed, whilst glucose had no effect. Regulation of endo-β-1,4-glucanase and β-glucosidase were compared.     

Expression of an Endo-β-1,4-glucanase Gene from Orpinomyces PC-2 in Pichia pastoris

The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.     

木聚糖酶最适pH值的研究

研究了以里氏木霉RutC - 3 0合成的木聚糖酶的最适pH值范围。结果表明 ,内切—木聚糖酶的最适pH值范围在 4.0～ 5 .0之间 ,β—木糖苷酶的最适pH值范围在 3 .0～ 4.0之间。研究还表明 ,与内切—木聚糖酶相比 ,pH值对β—木糖苷酶的影响更大。当pH值为 4.0时 ,酶水解总糖得率最高 ,适于制备木糖 ,而当pH值为 5 .0～ 6 .0时 ,较适于制备木低聚糖。     

Production of cellulases from coconut coir pith in solid state fermentation

Coconut coir pith, available in abundance especially in tropical countries, was studied as a substrate for the production of cellulase[1,4(1,3;1,4)-β-D -glucan 4-glucanohydrolase, EC 3.2.1.4] and β-D -glucosidase(β-D -glucoside glucohydrolase, EC 3.2.1.21) in solid state fermentation. The effects of fermentation time, nutrient level, substrate particle size and inoculum size have been examined for optimal production of these enzymes by the fungal strain Aspergillus niger NCIM 1005. The highest filter paper activity (FPA) of 4.11 IU g^?1, carboxyl methyl cellulose (CMCase) activity of 15·55 IU g^?1 and cellobiase activity of 9·31 IU g^?1 were obtained after 7 to 8 days of fermentation. Reese and Mandel's mineral solution in the substrate to mineral solution ratio of 1:10 (w/v) supported high cellulase and cellobiase activities. An inoculum size of 20–50% (v/v) based on the volume of mineral medium and substrate average particle size of 375 μm were optimum for enzyme production.     

Characterization of cellulase and xylanase activities of Clostridium celerecrescens

The production of cellulases and xylanase by Clostridium celerecrescens, a new anaerobic mesophilic cellulolytic bacterium, was studied using various substrates (cellobiose, xylan and cellulose Whatman CF-11). While both cellulase (β-1,4-D-glucan glucanohydrolase) and xylanase (β-1,4-xylan xylanohydrolase) were produced on cellulose, only the latter was produced when xylan was used as the sole carbon source. A weak p-nitrophenyl-β-D cellobiohydrolase activity was detected in the extracellular filtrates when using cellulose as a substrate. Otherwise, β-glucosidase (p-nitrophenyl-β-D-glucopyranosidase) was always found to be associated with the bacteria and reached its maximum levels of growth on cellobiose. In all cases, enzyme production showed a cell growth associated profile. Activities of these enzymes had their optimal values within the ranges of temperature and pH reported for the corresponding enzymes from similar anaerobic mesophilic microorganisms, although a relatively high optimum temperature, 55°C, was found for xylanase. All enzymes showed a 90% reduction of half-life time for each 8°C increment of temperature. A 50% inhibition of xylanase and β-cellobiohydrolase activity was observed, through a competitive mechanism, by xylose (0.677 mmol dm^?3) and cellobiose (28 mmol dm^?3) respectively.     

Purification and properties of β-amylase produced by bacillus polymyxa

1,4-α-D -glucan maltohydrolase (β-amylase, EC.3.2.1.2.) produced by Bacillus polymyxa was isolated and purified. Some of its properties were examined and compared with those of a typical plant β-amylase. Hydrolysis of periodate oxidised amylose demonstrated an exo mechanism of substrate attack similar to that of sweet potato β-amylase. The effect of sulphydryl reagents on enzyme activity was similar to that reported for plant β-amylases. Consistent with the observation that the enzyme has an exo mechanism of action, it also failed to degrade Schardinger cyclodextrins. These latter compounds acted as inhibitors of the enzyme. The optimum temperature for activity was 37 °C. The enzyme was quite stable at temperatures up to and including 37 °C; 90% of the original activity remained after storage at 37 °C for 6 days. However, the stability decreased rapidly when exposed to temperatures above 37 °C; only 20% of the activity remained after 1 h at 45 °C. The hydrolase exhibited a rather sharp optimum at pH 6.8 for stability at 37 °C. However, the enzyme was quite stable in the pH range 6.4–7.2 at 20 °C but it was shown to be less stable in acidic conditions than the corresponding plant enzymes.     

土壤杆菌-毕赤酵母耦合培养直接生产热凝胶低聚糖

为提高热凝胶低聚糖生产效率，构建了土壤杆菌?毕赤酵母耦合培养体系，其中土壤杆菌代谢产物热凝胶可被毕赤酵母分泌的内切-?-1,3-葡聚糖酶利用直接生产热凝胶低聚糖。用基于不同启动子(AOX1, GAP, FLD)调控的毕赤酵母重组菌株分泌表达内切-β-1,3-葡聚糖酶BGN13.1a，验证其均能有效水解热凝胶得到热凝胶低聚糖。在此基础上，选取GAP启动子调控的毕赤酵母工程菌与土壤杆菌耦合培养，通过设计两种物种之间的共生关系实现稳定共培养并生产聚合度为17?22的热凝胶低聚糖，产量为4.278 g/L。     

Thermal stabilization by polyols of β-xylanase from Bacillus amyloliquefaciens

Purified endo-β-1,4-xylanase of Bacillus amyloliquefaciens MIR 32 retained 100% of its activity after 4 days of incubation at 50°C. Sorbitol (400 mg cm⁻³) produced a 63-fold increase in the half-life of the enzyme at 65°C, which was only 29 min at this temperature in the absence of the polyol. This thermal stabilizing activity increased exponentially in respect to sorbitol concentration in the range 250–400 mg cm⁻³ and was dependent on the pH, showing a maximum at pH values between 5·25 and 8·0. The circular dichroism (CD) thermal scanning profile (50°C h⁻¹) at 224 nm showed that changes in the secondary structure of xylanase started at 65°C, while in the presence of sorbitol (400 mg cm⁻³) these modifications started at 80°C. This study indicated that sorbitol might be a valuable stabilizer for the use of β-xylanase from B. amyloliquefaciens at high temperatures. © 1998 SCI     

固定床反应器培养固定化大肠杆菌生产β-葡聚糖酶

以多孔陶瓷为载体吸附法固定重组大肠杆菌E. coli JM 109-pLF3表达胞外β-葡聚糖酶。考察了固定床间歇培养时循环流速和曝气量对发酵液酶活力的影响。当循环流速达到44.19 mL/min,曝气量达到0.6 mL/min时,培养48 h后,发酵液的酶活力达100.3 U/mL。固定化细胞具有良好的重复使用能力,在连续5批次实验中,培养48 h后的酶活力均在100 U/mL左右。固定床连续培养时,固定化细胞能够保持恒定的产酶效率,当稀释率为0.05 h－1时,发酵液中得到的酶活力为39.1 U/mL。     

超滤分离里氏木霉木聚糖酶的研究

研究了进料速度、操作压力、木聚糖酶活大小对内切-β-木聚糖酶和β-木糖苷酶超滤分离的影响。结果表明,用超滤的方法可以将内切-β-木聚糖酶和β-木糖苷酶很好地分离,内切-β-木聚糖酶活的收率随着进料速度的提高而增加,当进料速度从 300mL/min 提高到 450 mL/min 时,内切-β-木聚糖酶活的收率从 79.21%提高到 91.35%;内切-β-木聚糖酶活的收率随着操作压力的提高而降低,当操作压力从 4 kPa 提高到15 kPa 时,内切-β-木聚糖酶活的收率从 89.91%下降到 66.48%,而且操作压力越大越容易阻塞超滤膜;内切-β-木聚糖酶活的收率随着酶活的降低而增加,当总木聚糖酶活一定时,木聚糖酶活从 45.18 IU/mL 下降到 22.59 IU/mL 时,内切-β-木聚糖酶活的收率从 82.63%提高到 89.91%,木聚糖酶活较低时,酶液的体积增大,超滤时间过长,也会导致内切-β-木聚糖酶活收率降低。因此,在木聚糖酶活较低的情况下(酶活为22.59 IU/mL),温度为 25℃时,内切-β-木聚糖酶和β-木糖苷酶的适宜的分离条件为:压力为 4 kPa,进料速度为 450 mL/min,内切-β-木聚糖酶活的收率为 91.35%。     

The separation of pectinlyase from β-glucosidase in a commercial preparation

This paper describes a new, simple and inexpensive procedure for separating pectinlyase (PL, EC 4.2.2.3) from β-glucosidase (β-glu, EC 3.2.1.21), both of which have potential for use in the beverage processing industry. The method described here, which entails the treatment of crude preparations with bentonite (4% (w/v)) and the acidification of the resulting supernatant to pH 3·5, leads to the production of two enzymic solutions which contain PL and β-glu, respectively. In both solutions the amount of brown pigment is considerably less than in the crude mixture, and partial purification from extraneous proteins is also achieved.     

Stabilisation of β-glucosidase entrapped in alginate and polyacrylamide gels towards thermal and proteolytic deactivation

β-D -Glucosidase was immobilised by entrapment in two different matrices (calcium alginate and polyacrylamide gels), in order to compare how the immobilisation could stabilise the enzyme towards thermal and proteolytic deactivation. While the enzyme trapped in polyacrylamide gel showed an optimum temperature for activity at 10°C lower than that of the free enzyme, the optimal temperature after immobilisation in alginate beads was not altered (60°C). The immobilisation of enzyme in alginate beads caused a larger increase in the thermal stability than the entrapment in polyacrylamide gels. The stabilisation factors obtained as 55, 60 and 65°C for β-glucosidase immobilised in alginate and polyacrylamide gels were 2·03, 3·06, 2·19 and 2·04, 0·35, 1·01, respectively. In contrast, the β-glucosidase immobilised in polyacrylamide gels was more resist-ant in proteolysis than that trapped in alginate beads. © 1998 Society of Chemical Industry     

Characterization of extracellular thermostable xylanase from Chaetomium globosum

This paper deals with the characterization of extracellular thermostable xylanase produced by Chaetomium globosum, an ascomycete fungus. The crude extracellular enzyme was found to have temperature and pH optima of 60°C and 5·0 respectively. The enzyme hydrolyses xylan to a mixture of xylo-oligomers. Xylobiose and xylose are the major breakdown products.     

Direct ethanol production from rice straw by coculture with two high-performing fungi

To develop efficient and economical direct ethanol production from fine rice straw crashed mechanically, two high-performing fungi, which can secret hyperactive cellulases and/or ferment effectively various sugars, were selected from some strains belong to Mucor circinelloides preserved in our laboratory. The simultaneous saccharification and fermentation (SSF) by coculture with these fungi was investigated. The screening of high-performing fungi resulted in the selection of NBRC 4572 as an ethanol-producing fungus and NBRC 5398 as a cellulase-secreting fungus. The strain 4572 produced ethanol aerobically from glucose and xylose in high yields of 0.420 g/g at 36 h and 0.478 g/g at 60 h, respectively, but secreted fairly low cellulases. On the other hand, the strain 5398 also produced ethanol from glucose in yield of 0.340 g/g though it had a little growth in xylose culture. However, it secreted hyperactive cellulases that are essential for hydrolysis of rice straw in culture and the maximum activities of endo-β-glucanase and β-glucosidase were 2.11 U/L and 1.47 U/L, respectively. In SSF of rice straw by coculture with two fungi selected, the ethanol production reached 1.28 g/L after 96 h when the inoculation ratio of the strain 5398 to the strain 4572 was 9.     

Immobilization of β-glucosidase and its aroma-increasing effect on tea beverage

β-Glucosidase was effectively immobilized on alginate by the method of crosslinking–entrapment–crosslinking. After optimization of the immobilized conditions, the activity recovery of immobilized β-glucosidase achieved to 46.0%. The properties of immobilized β-glucosidase were investigated. Its optimum temperature was determined to be 45 °C, decreasing 10 °C compared with that of free enzyme, whereas the optimum pH did not change. The thermal and pH stabilities of immobilized β-glucosidase increased to some degree. The K_m value for immobilized β-glucosidase was estimated to be 1.97 × 10^?3 mol/L. The immobilized β-glucosidase was also applied to treat the tea beverage to investigate its aroma-increasing effect. The results showed that after treated with immobilized β-glucosidase, the total amount of essential oil in green tea, oolong tea and black tea increased by 20.69%, 10.30% and 6.79%, respectively. The storage stability and reusability of the immobilized β-glucosidase were improved significantly, with 73.3% activity retention after stored for 42 days and 93.6% residual activity after repeatedly used for 50 times.     

Purification and characterization of cellulases from Clostridium papyrosolvens

The extracellular cellulolytic enzyme complex from Clostridium papyrosolvens was isolated from a culture of this organism grown on filter paper. The complex showed xylanase, carboxymethyl cellulase (CMCase) and Avicelase (but not β-glucosidase) activities. Non-denaturing polyacrylamide gel electrophoresis (PAGE) revealed two dominant bands, sited at the origin (corresponding to a fraction of high molecular weight) and at 280 kDa, and eight other very weak bands in the 20–180 kDa MW range. Gel overlay techniques showed strong CMCase activity in the region comprised between the origin and 320 kDa and in three distinct sites in the 40–230 kDa region. Xylanase activities were detected as a continuous band almost covering all the track. SDS—PAGE gave a multiplicity of different intensity bands in the 20–130 kDa range. Cellulolytic activities (CMCase in the 85–90 kDa range) and hemicellulolytic activities (xylanase at approximately 95, 60, 42 and 32 kDa) were detected through application of the corresponding CMC and xylan overlay techniques. Though anion-exchange chromatography (DEAE-Sephadex A-50) and gel filtration (Biogel P-100) techniques did not permit a good separation of the different cellulolytic activities, an 11.3-fold increase of the Avicelase specific activity was achieved in a fraction containing 38.5% of the original total activity. Maximum enzymatic activities in crude preparations were observed at pH 5.4 and 50°C for xylanase and at pH 4.8 and 45°C for CMCase. The Michaelis constants for CMCase and xylanase were respectively: V_max = 9.1 μg glucose equivalents min^? ml^?, Km=3.3 g CMcellulose liter^? and V_max=44.5 μg xylose equiuulents min^? ml^? and K_m=2.7 g xylun liter^?. Both enzymes were inhibited by a competitive mechanism.     

Production of β-glucosidase by Aspergillus niger using carbon sources derived from agricultural wastes

The effects of various medium carbon sources and salt solutions on the production of β-glucosidase (βG, EC 3.2.1.21) by Aspergillus niger have been studied. β-Glucosidase productivity was found to be 50 times greater than that reported previously.¹ This higher productivity was achieved by employing a mutant strain of the organism, readily available and inexpensive carbon sources, such as cellulose and orange peel, and a simple nutrient salt solution.     

Production and properties of Aspergillus niger inulinase

An inulinase producing Aspergillus niger strain was isolated from Compositae rhizosphere soil samples. High inulinase levels were produced on a corn steep liquor (CSL)-maltose medium in the absence of inulin at 28°C within 110 h of fermentation. Media based on CSL-sucrose yielded high cell-bound inulinase activity; on inulin-based media the enzyme was mainly extracellularly produced. Both crude extra-and intracellular inulinase preparations displayed identical pH and temperature optima with maximal activity at pH 4.3–4.4 and at 55–56°C. These properties are favourable in view of large scale inulinase application for pure fructose production. High operation temperatures would avoid microbial contamination of reactors and would allow the use of high inulin-substrate concentrations, a limiting factor in obtaining high conversion ratios. The remarkably low pH optimum prevents colour formation and undesirable chemical side reactions. An advantageous low ratio of invertase to inulinase activity (S/I value) of 0.85 was found for the crude extracellular enzyme preparation. Crude inulin (chicory) extracts are hydrolysed faster than pure inulin. Apart from inulin (100% hydrolysis), sucrose (45%) and raffinose (20%) are also hydrolysed, and no liberation of oligomers or of sucrose from inulin was observed. These facts indicate that the A. niger enzyme is an exo-acting inulinase. The above characteristics make this A. niger inulinase an industrially attractive enzyme for the preparation of pure fructose from inulin-containing agricultural crops.     

Effects of pH and temperature on the continuous production of amylolytic enzymes by bacillus amyloliquefaciens MIR-41

Bacillus amyloliquefaciens MIR-41 produces three exocellular amylolytic enzymes: α-amylase, pullulanase and α-glucosidase. Physicochemical factors such as pH and/or temperature may affect the enzyme yield. Continuous culture at a dilution rate of 0.15 h^?1 demonstrated that the maximum production of α-amylase and α-glucosidase occurred at 36°C and pH 6.8 and of pullulanase at 44°C and pH 5.6. The results indicated that combinations of temperature and pH might be used for the production of crude extracts of α-amylase or pullulanase.     

The use of covalently immobilized mycelia to modify the β-glucanase system of Aspergillus vesicolor

Aspergillus vesicolor mycelial cells were covalently immobilized on a glutaraldehyde-ε-amino-caproyl-NH₂-Separon, and the constitutively-produced β-glucanase system and some properties of purified β-1,3-glucanase were compared with those of freely suspended cells. It was found that the mycelial immobilization modulates the composition of the β-glucanase system as well as stimulates the export of a functionally different β-1,3-glucanase.