Phototoxicity of protoporphyrin as related to its subcellular localization in mice livers after short-term feeding with griseofulvin.

The substrate specificities of three cellulases and a beta-glucosidase purified from Thermoascus aurantiacus were examined. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulase II and cellulase III, readily hydrolyzed the mixed beta-1,3; beta-1,6-polysaccharides such as carboxymethyl-pachyman, yeast glucan and laminarin. Both cellulase I and the beta-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenin (beta-1,4; beta-1,3) was degraded by all three cellulases. Cellulase II cannot degrade carboxymethyl-cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-beta-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalysed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of carboxymethyl-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length.     

里氏木霉GH61家族糖苷酶的高效表达及酶学特性研究

【目的】GH61家族糖苷水解酶具有葡聚糖氧化酶活性,通过对葡聚糖链的随机氧化而破坏木质纤维素的结晶结构,从而使木质纤维素容易被纤维素酶降解。重组表达、纯化获得里氏木霉的GH61家族糖苷水解酶(TrGH61,原名为EGⅣ),并研究其在纤维素酶水解木质纤维素中的作用。【方法】通过Overlap PCR将里氏木霉丙酮酸脱羧酶的启动子、纤维二糖水解酶cbh1的信号肽、EGⅣ基因和PDC终止子依次连接构建了里氏木霉的表达盒,通过该表达盒使TrGH61蛋白基因整合到里氏木霉的基因组DNA上进行同源表达。研究表达产物TrGH61的水解活性、与纤维素酶水解协同效应,以及TrGH61作为金属氧化酶的特性研究。【结果】在PDC启动子的作用下,TrGH61得到高效表达,摇瓶培养的表达量达到2.33 g/L。TrGH61有微弱的内切葡萄糖苷酶活性,比活力为0.02 IU/mg,但能显著提高纤维素酶水解稻草粉的活性,协同度最高可达1.998。低浓度的金属离子Cu2+、Co2+和还原性电子供体还原型谷胱甘肽、L-抗坏血酸、焦性没食子酸均能显著促进其水解效应。TrGH61能够降低稻草粉纤维素聚合度和结晶度。【结论】通过PDC启动子可以实现TrGH61蛋白高效组成型表达,TrGH61作为纤维素酶活性促进因子,通过破坏纤维素结晶结构作用机制协同增强纤维素酶水解木质纤维素。     

The Experimental Herbicide CGA 325′615 Inhibits Synthesis of Crystalline Cellulose and Causes Accumulation of Non-Crystalline β-1,4-Glucan Associated with CesA Protein

Developing cotton (Gossypium hirsutum) fibers, cultured in vitro with their associated ovules, were used to compare the effects of two herbicides that inhibit cellulose synthesis: 2,6-dichlorobenzonitrile (DCB) and an experimental thiatriazine-based herbicide, CGA 325'615. CGA 325'615 in nanomolar concentrations or DCB in micromolar concentrations causes inhibition of synthesis of crystalline cellulose. Unlike DCB, CGA 325'615 also causes concomitant accumulation of non-crystalline beta-1,4-glucan that can be at least partially solubilized from fiber walls with ammonium oxalate. The unusual solubility of this accumulated glucan may be explained by its strong association with protein. Treatment of the glucan fraction with protease changes its size distribution and leads to precipitation of the glucan. Treatment of the glucan fraction with cellulase digests the glucan and also releases protein that has been characterized as GhCesA-1 and GhCesA-2--proteins that are believed to represent the catalytic subunit of cellulose synthase. The fact that cellulase treatment is required to release this protein indicates an extremely tight association of the glucan with the CesA proteins. In addition, CGA 325'615, but not DCB, also causes accumulation of CesA protein and a membrane-associated cellulase in the membrane fraction of fibers. In addition to the effects of CGA 325'615 on levels of both of these proteins, the level of both also shows coordinate regulation during fiber development, further suggesting they are both important for cellulose synthesis. The accumulation of non-crystalline glucan caused by CGA 325'615 mimics the phenotype of the cellulose-deficient rsw1 mutant of Arabidopsis that also accumulates an apparently similar glucan (T. Arioli, L. Peng, A.S. Betzner, J. Burn, W. Wittke, W. Herth, C. Camilleri, H. Hofte, J. Plazinski, R. Birch et al. [1998] Science 279: 717).     

Optimization of enzyme complexes for lignocellulose hydrolysis

The ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and beta-glucosidase activity. Although the well-documented relief of product inhibition by beta-glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non-cellulosic polysaccharides. It is suggested that so-called "accessory" enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non-cellulosic polysaccharides that coat cellulose fibers. A high-throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a approximately twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis,supramolecular structure,and substrate accessibility

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF‐pretreated corn stover but reached only ～60% for the DA‐pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non‐cellulose accessibility to cellulase (NCAC) based on adsorption of a non‐hydrolytic recombinant protein TGC were measured for the first time. The COSLIF‐pretreated corn stover had a CAC of 11.57 m²/g, nearly twice that of the DA‐pretreated biomass (5.89 m²/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA‐ and COSLIF‐pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility. Biotechnol. Bioeng. 2009;103: 715–724. © 2009 Wiley Periodicals, Inc.     

Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus.

Three cellulases and a beta-glucosidase were purified from the culture filtrate of the thermophilic fungus Thermoascus aurantiacus. The isolated enzymes were all homogeneous on polyacrylamide-disc-gel electrophoresis. Data from chromatography on Bio-Gel P-60 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated mol.wts. of 87000 (beta-glucosidase), 78000 (cellulase I), 49000 (cellulase II) and 34000 (cellulase III); the carbohydrate contents of the enzymes were 33.0, 5.5, 2.6 and 1.8% (w/w) respectively. Although the three purified cellulases were active towards filter paper, only cellulases I and III were active towards CM(carboxymethyl)-cellulose. Cellulase I was also active towards yeast glucan. The Km and catalytic-centre-activity values for the enzymes were as follows; 0.52 mumol/ml and 6.5 X 10(4) for beta-glucosidase on p-nitrophenyl beta-D-glucoside, 3.9 mg/ml and 6.3 for cellulase I on CM-cellulose, 1.2 mg/ml and 1.1 for cellulase I on yeast glucan, 35.5 mg/ml and 0.34 for cellulase II on filter paper, and 1.9 mg/ml and 33 for cellulase III on CM-cellulose.     

Increasing cellulose accessibility is more important than removing lignin: A comparison of cellulose solvent‐based lignocellulose fractionation and soaking in aqueous ammonia

While many pretreatments attempt to improve the enzymatic digestibility of biomass by removing lignin, this study shows that improving the surface area accessible to cellulase is a more important factor for achieving a high sugar yield. Here we compared the pretreatment of switchgrass by two methods, cellulose solvent‐ and organic solvent‐based lignocellulose fractionation (COSLIF) and soaking in aqueous ammonia (SAA). Following pretreatment, enzymatic hydrolysis was conducted at two cellulase loadings, 15 filter paper units (FPU)/g glucan and 3 FPU/g glucan, with and without BSA blocking of lignin absorption sites. The hydrolysis results showed that the lignin remaining after SAA had a significant negative effect on cellulase performance, despite the high level of delignification achieved with this pretreatment. No negative effect due to lignin was detected for COSLIF‐treated substrate. SEM micrographs, XRD crystallinity measurements, and cellulose accessibility to cellulase (CAC) determinations confirmed that COSLIF fully disrupted the cell wall structure, resulting in a 16‐fold increase in CAC, while SAA caused a 1.4‐fold CAC increase. A surface plot relating the lignin removal, CAC, and digestibility of numerous samples (both pure cellulosic substrates and lignocellulosic materials pretreated by several methods) was also developed to better understand the relative impacts of delignification and CAC on glucan digestibility. Biotechnol. Bioeng. 2011; 108:22–30. © 2010 Wiley Periodicals, Inc.     

Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes

Soil metagenomes represent an unlimited resource for the discovery of novel biocatalysts from soil microorganisms. Three large-inserts metagenomic DNA libraries were constructed from different grassland soil samples and screened for genes conferring cellulase or xylanase activity. Function-driven screening identified a novel cellulase-encoding gene (cel01) and two xylanase-encoding genes (xyn01 and xyn02). From sequence and protein domain analyses, Cel01 (831 amino acids) belongs to glycoside hydrolase family 9 whereas Xyn01 (170 amino acids) and Xyn02 (255 amino acids) are members of glycoside hydrolase family 11. Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases. Both Xyn01 and Xyn02 were most active at 60°C with high activities from 4 to 10 and optimal at pH 7 (Xyn01) and pH 6 (Xyn02). The cellulase gene, cel01, was expressed in E. coli BL21 and the recombinant enzyme (91.9 kDa) was purified. Cel01 exhibited high activity with soluble cellulose substrates containing β-1,4-linkages. Activity with microcrystalline cellulose was not detected. These data, together with the analysis of the degradation profiles of carboxymethyl cellulose and barley glucan indicated that Cel01 is an endo 1,4-β-glucanase. Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.     

Stimulatory effect of oxidized cellulose on cellulase formation by Trichoderma reesei

Crystalline cellulase has been electrochemically oxidized to yield preparations containing various different percentages of oxidized end-groups. These celluloses have been used as carbon sources for growth and cellulase production by Trichoderma reesei . A low content of oxidized end groups in the celluloses (0.1–0.65%) stimulated cellulase production but not growth, whereas higher contents (> 1%) where inhibitory to both. The cellulolytic enzyme system secreted under stimulated conditions contained the same proportion of individual cellulase enzymes (cellobiohydrolase I and II, endoglucanase I) as the control, indicating a general stimulatory effect of oxidized cellulose. Activity of cellulases against oxidized celluloses in vitro was not stimulated, and only slightly inhibitory at high degrees of oxidation. The data support a potential role of cellulose oxidation in regulating cellulase formation by T. reesei .     

Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass

The switchgrass (SG) samples pretreated by cellulose solvent‐ and organic solvent‐based lignocellulose fractionation were characterized by enzymatic hydrolysis, substrate accessibility assay, scanning electron microscopy, X‐ray diffraction (XRD), cross polarization/magic angle spinning (CP/MAS) ¹³C nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). Glucan digestibility of the pretreated SG was 89% at hour 36 at one filter paper unit of cellulase per gram of glucan. Crystallinity index (CrI) of pure cellulosic materials and SG before and after cellulose solvent‐based pretreatment were determined by XRD and NMR. CrI values varied greatly depending on measurement techniques, calculation approaches, and sample drying conditions, suggesting that the effects of CrI data obtained from dried samples on enzymatic hydrolysis of hydrated cellulosic materials should be interpreted with caution. Fast hydrolysis rates and high glucan digestibilities for pretreated SG were mainly attributed to a 16.3‐fold increase in cellulose accessibility to cellulase from 0.49 to 8.0 m²/g biomass, because the highly ordered hydrogen‐bonding networks in cellulose fibers of biomass were broken through cellulose dissolution in a cellulose solvent, as evidenced by CP/MAS ¹³C‐NMR and FTIR. Biotechnol. Bioeng. 2011; 108:521–529. © 2010 Wiley Periodicals, Inc.     

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and β-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase–xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Enzymatic digestion of liquid hot water pretreated hybrid poplar

Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200^oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot‐washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot‐washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar‐to‐ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.     

Mild pretreatment of yellow poplar biomass using sequential dilute acid and enzymatically-generated peracetic acid to enhance cellulase accessibility

Biomass contains cellulose, xylan and lignin in a complex interwoven structure that hinders enzymatic hydrolysis of the cellulose. To separate these components in yellow poplar biomass, we sequentially pretreated with dilute sulfuric acid and enzymatically-generated peracetic acid. In the first step, the dilute acid with microwave heating (140°C, 5 min) hydrolyzed 90% of xylan. The xylose yield in hydrolysate after dilute acid pretreatment was 83.1%. In the second step, peracetic acid (60°C, 6 h) removed up to 80% of lignin. This sequential pretreatment fractionated biomass into xylan and lignin, leaving a solid residue enriched in cellulose (~80%). The sequential pretreatment enhanced enzymatic digestibility of the cellulase by removal of the other components in biomass. The glucose yield after enzymatic hydrolysis was 90.5% at a low cellulase loading (5 FPU/g of glucan), which is 1.6 and 18 times higher than for dilute acid-pretreated biomass and raw biomass, respectively. This novel sequential pretreatment with dilute acid and peracetic acid efficiently separates the three major components of yellow poplar biomass, and reduces the amount of cellulase needed.     

Optimization of pH controlled liquid hot water pretreatment of corn stover

Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 degrees C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.     

Characterization of exopolysaccharide (EPS) produced by Weissella hellenica SKkimchi3 isolated from kimchi

Weissella hellenica SKkimchi3 produces the higher exopolysaccharide (EPS) on sucrose than lactose, glucose, and fructose at pH 5 and 20°C. Sucrose was exclusively used to cultivate SKkimchi3 in all experiments base on the EPS production tests. The molecular mass of EPS, as determined by gel permeation chroma-tography, was 203,000. ¹H and ¹³C NMR analysis indicated that the identity of EPS may be a glucan. When EPS, starch, and cellulose was treated with a-amylase, glucoamylase, glucosidase, and cellulase, glucose was produced from starch and cellulose but was not produced from EPS. Based on HPLC analysis, elemental analysis, ¹H and ¹³C NMR analysis, and enzymatic hydrolysis tests, EPS was estimated to be a glucan. EPS suspension was not precipitated even by centrifugation at 10,000×g for 60 min, and EPS made the fermented milk and bacterial culture viscous.