纤维素酶的二步分离纯化新工艺

 以普通定性滤纸为底物 ,经碱处理后 ,研究其对纤维素酶的亲和吸附作用。结果表明 ,普通定性滤纸对纤维素酶具有比较强的特异性吸附作用 ,能够从粗酶液中分离出纤维素酶 ,再经POROS 2 0HQ阴离子交换柱纯化后即可得到电泳纯的纤维素酶。该法大大简化了传统的纤维素酶纯化工艺 ,所得的纤维素酶活力极高 ,比活达 35 0U/mg以上 ,滤纸一步吸附后纤维素酶的纯化倍数为 9 5 5 ,活性回收率在 10 %左右。纯化后的纤维素酶为内切 β 葡聚糖酶 ,相对分子质量为 6 0 0 0 0 ,最佳 pH为 4 0 ,最佳温度为 70℃。     

Co-immobilization Mechanism of Cellulase and Xylanase on a Reversibly Soluble Polymer

Cellulase and xylanase from Trichoderma reesei were immobilized simultaneously on Eudragit L-100, a reversibly soluble polymer. The effects of polymer concentration and polymer precipitation pH on enzyme activity recovery were investigated at an enzyme complex concentration of 1%. The immobilization mechanism of cellulase and xylanase on the polymer was discussed. An activity recovery of 75% and 59% was obtained for the cellulase and the xylanase, respectively, under the condition of a polymer concentration at 2% and a polymer precipitation pH at 4.0. Most zymoproteins might be connected to the polymer by electrostatic attraction in a medium of pH 4.8. In addition, the covalent coupling between the zymoproteins and the polymer was demonstrated by the infrared spectrograms. It was suggested that dehydration–condensation reaction occurred between the zymoproteins and the polymer during the immobilization.     

A Novel Stepwise Recovery Strategy of Cellulase Adsorbed to the Residual Substrate after Hydrolysis of Steam Exploded Wheat Straw

Cellulase distribution between residual substrate and supernatant in the process of enzymatic hydrolysis of steam-exploded wheat straw was investigated. Subsequently, a novel stepwise recovery strategy with three successive steps was adopted to recover cellulase adsorbed to the residual substrate. The results showed that cellulase protein in the supernatant increased as the hydrolysis time increased. When hydrolysis ended, the cellulase remaining on the residual substrate accounted for 33–42% of the original added cellulase according to the different cellulase loading. To obtain the maximum cellulase recovery rate, the residual substrate was dealt with in three successive steps: washed with sodium acetate buffer (step 1), shaken with sodium acetate buffer (step 2), and then treated with 0.0015 mol/L, pH 10 Ca(OH)₂ (step 3). The total cellulase protein recovered by the three steps reached 96.70–98.14%. The enzyme activity of cellulase recovered by the first two steps was kept well. The ratios of the specific activity between the recovered cellulase and the original were 89–96%, which was by far higher than that using step 3 (the value was 48% ∼ 56%).     

Partial purification, and some properties and reactivities of cetraxate benzyl ester hydrochloride-hydrolyzing enzyme

Debenzylating enzyme from Aspergillus niger enzyme (commercial crude cellulase) catalyzes the hydrolysis of cetraxate benzyl ester hydrochloride (2), a precursor of the antiulcer agent (1). The enzyme was highly purified by three kinds of chromatographies (hydrophobic, ion exchange, gel filtration) with a recovery of 36%. The content of the debenzylating enzyme was about 0.1% in the crude cellulase, but the enzyme showed no cellulase activity. The purified enzyme was inactivated by Hg2+, and diisopropyl phosphorofluoridate (DFP). It was a monomer with a molecular weight of about 35,000, and its isoelectric point was estimated to be 5.3. It showed a debenzylating activity for the phenylpropionic acid benzyl ester moiety of various benzyl ester derivatives, and the benzyl ester of phenylalanine or that of tyrosine was also well hydrolyzed.     

Purification of phospholipase D by two-phase affinity extraction

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.     

Purification of cellulases from Streptomyces strain A20 by electroendosmotic preparative electrophoresis

Five cellulase components were identified and purified in one step from Streptomyces strain A20 using electroendosmotic preparative electrophoresis. By this procedure up to 18 mg of protein mixture could be loaded on the column, with an estimated recovery of 60-70% of total activity; activity and protein recovery could be estimated 32% and 47% respectively, if only activity peaks were considered. In comparison to other purification methods, this technique results in high protein recovery and resolution of applied samples.     

利用高效弱阴离子交换色谱法纯化超氧化物歧化酶

利用高软弱阴离子交换色谱法简化了超氧化物歧化酶（ＳＯＤ）的纯化手续。在丙酮沉淀后,仅用一步色谱分离就能使来自牛血的Ｃｕ,Ｚｎ－ＳＯＤ达到电泳纯,活性回收率为８６．４％,比活为７７１１Ｕ／ｍｇ,纯化倍数提高至５２倍。此外,详尽讨论了色谱分离的条件。     

Efficient Production of γ-GABA Using Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ～47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg^?1. The purified cellulase retained its activity over a wide range of temperature (50–70 °C) and pH (3–7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.     

Studies on the chromatographic fractionation of Trichoderma reesei cellulases by hydrophobic interaction

This work reports new studies on cellulases fractionation by hydrophobic interaction chromatography. The purification procedure for the Trichoderma reesei cellulase complex consists of gel permeation chromatography on Sephadex G-25M followed by an ultrafiltration step. The concentrated enzyme solution was then fractionated on Sepharose CL-6B modified by covalent immobilization of 1,4-butanediol diglycidyl ether. The influence of the mobile phase composition on the chromatographic behaviour of the T. reesei cellulase complex was investigated. By using 13% (w/v) ammonium sulphate in eluent buffer, a selective separation of beta-glucosidase with a two-fold increase in specific activity and a recovery of 60% cellobiase activity were obtained. Other commercial hydrophobic supports (octyl- and phenyl-Sepharose) were also tested and compared under the same conditions.     

Purification of a rat liver phenol sulphotransferase (P-STG) with the aid of guanidine hydrochloride treatment.

An isoenzyme of phenol sulphotransferase, designated P-STG, was purified 157-fold from male rat liver cytosol by diethylaminoethyl-cellulose (DEAE-cellulose) and agarose-hexane-adenosine-3',5'-bisphosphate affinity chromatography. The P-STG fraction obtained after DEAE-cellulose chromatography rapidly lost its activity during storage at 4 degrees C, however, the activity was recovered by the addition of 1.6 M guanidine hydrochloride (Gndn HCl) followed by dialysis. Gndn HCl also substantially improved the yield of P-STG in a subsequent purification step using affinity chromatography, while the specific activity of the purified P-STG was not changed by Gndn HCl treatment. It is possible that the Gndn HCl treatment caused P-STG recovery from an inactivated to an active form rather than reactivating it for increased activity. Purified P-STG is a homodimer with a native molecular mass of 67 kDa; the subunit molecular mass is 35 kDa. Immunoblot analysis carried out with antibodies raised against the purified enzyme indicated that male rat liver contains a higher level of the enzyme than female rat liver. This enzyme is also expressed in the kidney and the stomach. P-STG reaches maximum activity when 1-naphthol, 2-naphthol and 4-nitrophenol are used as substrates at pH 5.5. Using dopamine as a substrate the pH optimum is about 9.0. P-STG activity is markedly inhibited by the addition of sodium chloride to the reaction mixture.     

Optimization,Purification, and Characterization of Extracellular Mesophilic Alkaline Cellulase from Sponge-Associated <Emphasis Type="Italic">Marinobacter</Emphasis> sp. MSI032

Marinobacter sp. (MSI032) isolated from the marine sponge Dendrilla nigra was optimized for the production of extracellular cellulolytic enzyme (CMCase) by submerged fermentation. Initial experiments showed that the culture medium containing 1% maltose as carbon source and 1% peptone and casein as nitrogen source supported maximal enzyme production at 27 °C and at a pH of 9.0. Further optimization carried out showed the maximal enzyme production was supported by the presence of 2% NaCl and 10 mM Zn²⁺ ions in the production media. The production of enzyme cellulase occurred at 48 h of incubation which proved the importance of this strain for cellulase production in large scale. Further, the enzyme was purified to 12.5-fold with a 37% yield and a specific activity of 2,548.75 U/mg. The purified enzyme displayed maximum activity at mesophilic temperature (27–35 °C) and at a broad pH range with optimal activity at pH 9.0. The purified enzyme was stable even at a higher alkaline pH of 12.0 which is greater than the pH stability that has not been reported in any of the cellulolytic isolates studied so far. Thus, from the present study, it is crucial that, instead of exploring the thermophilic resource that is limited in natural environments, the mesophilic bacteria that occurs commonly in nature can be added up to the database of cellulolytic bacteria. Thus, it is possible that a wide diversity of mesophilic bacteria associated with marine sponges opens up a new doorstep for the degradation of cellulosic waste material for the production of liquid fuels. This is the first report elucidating the prospects of sponge-associated marine bacterium for the production of extracellular alkaline cellulase.     

Enzyme recovery and recycling following hydrolysis of ammonia fiber explosion-treated corn stover

Both cellulase and cellobiase can be effectively recovered from hydrolyzed biomass using an ultrafiltration recovery method. Recovery of cellulase ranged from 60 to 66.6% and for cellobiase from 76.4 to 88%. Economic analysis shows that cost savings gained by enzyme recycling are sensitive to enzyme pricing and loading. At the demonstrated recovery of 60% and current loading of 15 Filter paper units of cellulase/g of glucan, enzyme recycling is expected to generate a cost savings of approx 15%. If recovery efficiency can be improved to 70%, the savings will increase to >25%, and at 90% recovery the savings will be 50%.     

Reversibly soluble macroaffinity ligand in aqueous two-phase separation of enzymes

Use of alginate as a free bioligand incorporated in an aqueous two-phase system of polyethylene glycol 6000-salt resulted in considerable purification of wheat germ alpha-amylase and sweet potato beta-amylase from their crude extracts. The elution of the enzyme from the free bioligand was facilitated by exploiting the fact that alginate can be reversibly precipitated in the presence of Ca2+. alpha-Amylase could be purified 42-fold with 92% activity recovery. beta-Amylase on the other hand could be purified 43-fold with 90% recovery. Both purified enzymes showed a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

Production,purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60 degrees C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0, retaining >80% of its original activity within this range. Half-lives of 150 min at 50 degrees C and 6.5 min at 60 degrees C were found. K(m) and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birchwood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-D-glucuronoxylan with a K(m) of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Immobilization of cellulase in nanofibrous PVA membranes by electrospinning

Electrospinning is a nanofiber-forming process by which either polymer solution or melt is charged to high voltages. With high specific surface area and porous structure, electrospun fibrous membranes are excellent candidates for immobilization of enzymes. In this paper, immobilization of cellulase in nanofibrous poly(vinyl alcohol) (PVA) membranes was studied by electrospinning. PVA and cellulase were dissolved together in an acetic acid buffer (pH 4.6) and electrospun into nanofibers with diameter of around 200 nm. The nanofibrous membranes were crosslinked by glutaraldehyde vapor and examined catalytic efficiency for biotransformations. The activity of immobilized cellulase in PVA nanofibers was over 65% of that of the free enzyme. Nanofibers were superior to casting films from the same solution for immobilization of cellulase. The activity of immobilized cellulase descended with ascending in enzyme loading efficiency and crosslinking time, which retained 36% its initial activity after six cycles of reuse.     

Rapid,High-Yield Purification of Rat Liver Glutathione Peroxidase by High Performance Liquid Chromatography

Abstract

Glutathione peroxidase (GSH:H₂O₂ oxidoreductase, EC 1.11.1.9) was purified 3500-fold from rat liver with a yield of 42% using high performance liquid chromatography. The crucial purification step was size-exclusion chromatography on a Spherogel TSK-3000SW column, and the purified enzyme eluted as a single peak. The enzyme stained as a single band following SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be 105,000, and the subunit molecular weight determined by SDS-gel electrophoresis was 25,000. Polyacrylamide gel electrophoresis indicated five bands of protein with a broad of enzymatic activity. Isoelectric focusing resulted in a peak of enzymatic activity at pH 6.9 with a shoulder at pH 7.3. The specific activity of the purified enzyme was 1,100 μmol of NADPH oxidized per minute per milligram of protein.     

Functional Stabilization of Cellulase from Aspergillus niger by Conjugation with Dextran-aldehyde

The covalent conjugates of cellulase from Aspergillus niger were prepared with various molar ratios by using dextran. The conjugate (n_E/n_D: 1/5) showed higher activity than purified enzyme at all temperatures after 1 h of incubation and its activity could also be measured at higher temperature. Also, this conjugate lost only 60% activity in 2 h at 70°C in comparison to the purified enzyme, which lost all its activity. In addition, conjugation protected cellulase against denaturation in the presence of sodium dodecylsulfate (residual activity of about 80%) and inactivation by air bubbles (residual activity of about 50% for 4 h).     

kappa-Carrageenan as a new smart macroaffinity ligand for the purification of pullulanase

kappa-Carrageenan is a polysaccharide from red seaweed which gets precipitated by K+ ions and dissolves again in water. This smart, K(+)-responsive polymer was found to selectively bind pullulanase activity from Bacillus acidopullulyticus. Gel filtration on Sephadex G-200 showed the formation of the polymer-pullulanase complex at the pre-precipitation stage. On the other hand, phospholipase D, an enzyme which did not co-precipitate with kappa-carrageenan, did not form any complex with the polymer. Thus, K+ ions could be used to selectively precipitate the pullulanase activity. Then, 92% enzyme activity could be eluted with 1 M maltose solution. The single step protocol resulted in 50-fold purification, with a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

Purification and properties of membrane-bound coupling factor-latent ATPase from Mycobacterium phlei.

The membrane bound coupling factor-latent ATPase was solubilized from the membrane vesicles of Mycobacterium phlei by using 0.25 M sucrose or low ionic strength buffer. Purification of the solubilized enzyme by use of Sepharose-ADP conjugate gel yielded a homogenous preparation of latent ATPase which was purified about 216-fold in a single step with an 84% yield. The enzyme exhibits a specific activity of 39 mumoles of ATP hydrolyzed per min per mg protein. The purified enzyme exhibits coupling factor activity. Electrophoresis in two dissociating solvent systems indicates that the enzyme contains at least three major polypeptides of molecular weights 56,000, 51,000, and 46,000 daltons, and two minor polypeptides of 30,000 and 17,000 daltons. Equilibrium binding studies of ADP with purified coupling factor-latent ATPase reveal the presence of two nucleotide binding sites per molecule with an apparent Ka of 8.1 X 10(-5) M. By use of affinity chromatography, another latent ATPase has been isolated from the solubilized enzyme, which does not exhibit coupling factor activity.     

One‐step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X‐100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio‐active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.