Cellulolytic properties of Chaetomium crispatum

The cellulolytic properties of a Chaetomium crispatum strain were investigated. The cellulolytic enzyme complex i.e.: exo-1,4-β-glucosidase (EC 3.2.1.74); endo-1,4-β-glucanase (EC 3.2.1.4.) and β-glucosidase or cellobiase (EC 3.2.1.21) displayed optimal activity at pH 5.0 and 25°C. Although carboxymethyl-celluloses are the usual pseudo-substrates for this enzyme complex, those with a high degree of substitution gave rise to poor growth and low cellulase activity. Insoluble crude cellulosics such as newsprint, recycled paper, rice and flax straw were substantially solubilised at 28°C within 3–5 days of fermentation. A study of the cellulase-complex formation during the growth cycle revealed that β-glucosidase was produced mainly intracellularly in the early exponential phase, while the overall exo-1,4-β-glucosidase and endo-1,4-β-glucanase formation gradually increased during the total fermentation cycle. The mycelial protein of Chaetomium crispatum grown on crude cellulosics displayed a favourable amino acid pattern, indicating its potential value as a source of single cell protein (SCP).     

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.     

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.     

Some properties of β-D-Glucosidase from Trichoderma harzianum C1R1

β-D-Glucosidase from Trichoderma harzianum C₁R₁ consists of several isocomponents having isoelectric points in the pH range of 4.85-7.50. All the components exhibit both cellobiase and 4-nitrophenyl β-D-glucosidase (4NPGase) activity. The enzyme affinity for cellobiose (K_m = 3.92 mmol dm^?3) is 14.5 times weaker than for 4NPG (K_m = 0.27 mmol dm^?3). The hydrolysis of both substrates is competitively inhibited by glucose, the inhibition of 4NPG hydrolysis (K₁ = 2.00 mmol dm^?3) being about 4.2 times stronger compared to the hydrolysis of cellobiose (K₁ = 8.43 mmol dm^?3). The 4NPG hydrolysis is also competitively inhibited by the presence of cellobiose and D-glucono-1,5-lactone (K_i(cellobiose) = 5.00 mmol dm^?3; K_{i(D-glucono-1,5-lactone}) = 22 μmol dm^?3). The optimal hydrolysis conditions are the same for both substrates (pH 4.5,55° C). The half-lives of thermal inactivation at 61° C are 27 and 10min for cellobiase and 4NPGase, respectively.     

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.     

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     

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.     

运用试验设计方法提高胞外杂合b-葡聚糖酶在重组大肠杆菌中的表达

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L^-1. After optimization of the medium, 297.71U·ml^-1 of β-glucanase activity in the medium and 352350U·g^-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.     

Enhancement of immobilized enzyme activity by pretreatment of β-glucosidase with cellobiose and glucose

In this study, β-glucosidase from Aspergillus niger was pretreated with cellobiose and glucose to prevent loss of enzyme activity, and pretreated β-glucosidase was immobilized on silica gel as a carrier by covalent binding. To enhance the activity of immobilized β-glucosidase, the effects of substrate concentration and reaction conditions, including temperature, time, and agitation speed, were investigated. The optimal concentrations of cellobiose and glucose, temperature, time, and agitation speed were determined to be 0.02 M, 40 °C, 20 min, and 130 rpm, respectively. The activity of immobilized β-glucosidase after pretreatment was increased to about 176% of that of non-pretreated β-glucosidase. In addition, the optimal pH and temperature of the non-pretreated and pretreated immobilized β-glucosidases were both pH 5.5 and 65 °C, respectively. Moreover, the immobilized β-glucosidases were used repeatedly 20 times, and the enzyme activities were maintained at levels higher than 80% of their initial activities.     

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     

Comparison of β-galactosidase immobilization by entrapment in and adsorption on poly(2-hydroxyethylmethacrylate) membranes

β-Galactosidase was immobilized in/on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two different methods: adsorption on Cibacron F3GA derivatized pHEMA membranes (pHEMA-CB), and entrapment in the bulk of the pHEMA membranes. The maximum β-galactosidase adsorption on pHEMA-CB membranes was obtained as 95·6μgcm^-2 in 2·0mgcm^-3 enzyme solution. The adsorption phenomena appeared to follow a typical Langmuir isotherm. In the entrapment, an increase in β-galactosidase loading resulted in a consistent increase in membrane activity from 3·3×10^-2 to 17·8×10^-2Ucm^-2 pHEMA membranes. The K_m values for both immobilized β-galactosidase (adsorbed 0·32mM and entrapped 0·81mM ) were higher than that of the free enzyme (0·26mM ). The optimum reaction temperature of the adsorbed enzyme was 5°C higher than that of both the free and the entrapped enzyme. The optimum reaction pH was 7·5 for free and both immobilized preparations. After 15 successive uses the retained activity of the adsorbed and the entrapped enzymes was 80% and 95%, respectively. The storage stability of the enzyme was found to increase upon immobilization. ©1997 SCI     

Ruthenium‐Catalyzed Oxidation of the Porphyrin β,β′‐Pyrrolic Ring: A General and Efficient Approach to Porpholactones

We describe an efficient ruthenium‐catalyzed oxidation of the β,β′‐pyrrolic ring on the porphyrin periphery. Through the conversion of a β,β′‐double bond to a lactone moiety, the direct preparation of porpholactones from porphyrins is achieved, which previously suffered from needing toxic reagents, multiple synthetic steps and low yields. The generality of this method has been investigated with various porphyrins with different electronic and steric effects, even some metalloporphyrins, and so represents a general and efficient approach for the synthesis of the intriguing porpholactone derivatives.     

The Tertiary Amine Nitrogen Atom of Piperazine Sulfonamides as a Novel Determinant of Potent and Selective β3‐Adrenoceptor Agonists

Novel compounds were prepared in fair to good yields as human β₃‐adrenoceptor (β₃‐AR) agonists. In particular, aryloxypropanolamines 7 a – d (EC₅₀=0.57–2.1 nM ) and arylethanolamines 12 a , b , e (EC₅₀=6.38–19.4 nM ) were designed to explore the effects of modifications at the right‐hand side of these molecules on their activity as β₃‐AR agonists. Piperidine sulfonamides 15 a – c , e – g (EC₅₀=6.1–36.2 nM ) and piperazine sulfonamide derivatives 20 – 29 (EC₅₀=1.79–49.3 nM ) were examined as compounds bearing a non‐aromatic linker on the right‐ and left‐hand sides of the molecules. Some piperazine sulfonamides were found to be potent and selective β₃‐AR agonists, even if the amine nitrogen atom is tertiary and not secondary, as is the case for all β₃‐AR agonists reported so far. (S)‐3‐{4‐{N‐{4‐{2‐[2‐Hydroxy‐3‐(4‐hydroxyphenoxy)propylamino]ethyl}phenyl}sulfamoyl}phenoxy}propanoic acid ( 7 d ; EC₅₀=0.57 nM ), (R)‐N‐{4‐[2‐(2‐hydroxy‐2‐phenylethylamino)ethyl]phenyl}‐4‐(3‐octylureido)benzenesulfonamide ( 12 e ; EC₅₀=6.38 nM ), (R)‐2‐[1‐(4‐methoxyphenylsulfonyl)piperidin‐4‐ylamino]‐1‐phenylethanol ( 15 f ; EC₅₀=6.1 nM ), and (S)‐4‐{2‐hydroxy‐3‐[4‐(4‐methoxyphenylsulfonyl)piperazin‐1‐yl]propoxy}phenol ( 25 ; EC₅₀=1.79 nM ) were found to be the most potent β₃‐AR agonists of the aryloxypropanolamine, arylethanolamine, piperidine sulfonamide, and piperazine sulfonamide classes, respectively. The two most potent compounds were identified as possible candidates for further development of β₃‐AR agonists useful in the treatment of β₃‐AR‐mediated pathological conditions.     

A Combined High‐Resolution Mass Spectrometric and in silico Approach for the Characterisation of Small Ligands of β2‐Microglobulin

β₂‐Microglobulin (β₂‐m) is a protein responsible for a severe complication of long‐term hemodialysis, known as dialysis‐related amyloidosis, in which initial β₂‐m misfolding leads to amyloid fibril deposition, mainly in the skeletal tissue. Whereas much attention is paid to understanding the complex mechanism of amyloid formation, the evaluation of small molecules that may bind β₂‐m and possibly inhibit the aggregation process is still largely unexplored mainly because the protein lacks a specific active site. Based on our previous findings, we selected a pilot set of sulfonated molecules that are known to either bind or not to the protein, including binders that are anti‐amyloidogenic. We show how a complementary approach, using high‐resolution mass spectrometry and in silico studies, can offer rapid and precise information on affinity, as well as insight into the structural requisites that favour or disfavour the inhibitory activity. Overall, this approach can be used for predictive purposes and for a rapid screening of fibrillogenesis inhibitors.     

Use of Beta Cyclodextrin to Remove Cholesterol and Increase Astaxanthin Content in Shrimp Oil

Shrimp oil is extracted from shrimp (Litopenaeus vannamei) cephalothorax and subjected to the removal of cholesterol by β‐cyclodextrin (βCD). Different oil/βCD ratios (1:2, 1:3, and 1:4, w/w) and homogenization times (1, 10, and 20 min) are used. Cholesterol deduction is attained with increasing βCD levels and homogenization time. Astaxanthin content is augmented, while cholesterol concentration is reduced. Nevertheless, oil yield and astaxanthin concentration of treated oil are decreased as βCD levels are increased. To increase the oil yield, the used βCD is further extracted for three times with ethyl acetate at 1:10 (w/v) ratio, in which yield is increased from 44.6% to 64%. Cholesterol removal of 95% is obtained, while astaxanthin content is increased. Lipid oxidation is lowered as indicated by the lower TOTOX value, peroxide value, thiobarbituric acid reactive substances, and p‐anisidine value. However, lipid hydrolysis is slightly increased after treatment. Volatiles, especially aldehydes and alcohols, are decreased after treatment. FTIR spectra confirm the removal of phospholipid, which might be associated with the decreased oil yield after treatment. With the developed process, total fatty acid is increased by 15.6%, in which monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) are augmented. βCD could remove cholesterol, increase astaxanthin and fatty acid content. Practical Applications: Shrimp oil has been known to be a rich source of astaxanthin and PUFAs with health benefit. However, it also contains cholesterol, which can be a drawback for consumption as the supplement. The removal of cholesterol, while maintaining PUFA and astaxanthin could pave a way for promoting the intake of shrimp oil. Use of βCD for oil treatment with subsequent extraction of remaining oil in the used βCD could be implemented with ease. Another advantage of the developed process is to increase both fatty acid and astaxanthin contents in the resulting oil. As a consequence, shrimp oil with lowered cholesterol can be directly used as food ingredient and also for neutraceutical purpose.     

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 effect of β-dicalcium silicate, α-monocalcium silicate and calcium hydroxide upon the removal of lead (pb2+) ions from aqueous solution

A study was conducted of the influence of pH, initial reactant concentration and reaction time upon the rate of removal of lead ions from solutions of lead nitrate. Three different reactants were used: β-dicalcium silicate (β-C₂S)*, α-monocalcium silicate (α-CS), in the form of finely ground clinkers, and commercial calcium hydroxide. The initial concentration of lead in solution was varied between 5 and 1000 mg cm^-3 Pb²⁺ for different additions of reactant. For each concentration of lead ion the effect of time of shaking on the removal of lead was followed by the determination of residual lead concentration in the filtrate. Lead was reduced to low levels by all the precipitants. The precipitated reaction products were identified by X-ray diffraction techniques, electron diffraction analysis and infrared spectroscopy. The analysis characterised the products of the precipitation reactions as basic lead carbonate, basic lead nitrates and lead hydroxide.     

Fabrication of citric acid crosslinked β‐cyclodextrin/hydroxyethylcellulose hydrogel films for controlled delivery of poorly soluble drugs

β‐cyclodextrin grafted hydroxyethylcellulose (βCD‐g‐HEC) hydrogel films were prepared for the controlled release of poorly soluble model drug (ketoconazole) using citric acid as crosslinking agent. The active βCD and carboxyl content of the hydrogel films were determined by phenolphthalein assay and acid–base titration. The films were characterized by solid state ¹³C NMR, ATR–FTIR, thermogravimetric analysis, and differential scanning calorimetric, and analyzed for tensile strength, swelling ratio, drug loading, release, hemocompatibility, in vitro cytotoxicity, and implantation test. An increase in the concentration of βCD in feed increased the active βCD content of the hydrogel films but reduced their extent of interpolymer crosslinking. The βCD‐g‐HEC hydrogel films with high active βCD content showed maximum drug loading whereas those with high crosslinking density were capable of controlling the drug release for long duration. Hemolysis assay and in vitro cytotoxicity study revealed the biocompatible nature of the hydrogel films whereas implantation test indicated their minimal inflammatory effect. From the overall results, βCD‐g‐HEC hydrogel films were found to be better alternative to the previously reported βCD‐HPMC and βCD‐CMC hydrogel films for enhanced loading and long‐term release, respectively, of the poorly soluble drugs. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46452.     

One‐Pot Synthesis of Fluorovinyl Acetates and β,β‐Difluoro Carboxylates from a Hypervalent Iodine and Hydrogen Fluoride‐Based Fluorination Reagent

A new strategy for the synthesis of fluorovinyl acetates and β,β‐difluoro carboxylates using a hypervalent iodine and hydrogen fluoride‐based fluorination reagent is established through a silver‐catalyzed one‐pot reaction involving formation of carbon‐oxygen and carbon‐nitrogen bonds. The generated fluoroalkenes substituted with the carbon‐oxygen and carbon‐nitrogen bond on the β‐position have the potential to be transformed into various products which would be difficult to obtain via direct fluorination.

     

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.