Thermostable Mutants of Glycoside Hydrolase Family 6 Cellobiohydrolase from the Basidiomycete Phanerochaete chrysosporium

Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass. However, while the catalytic rate of enzymes generally becomes faster at higher temperatures, Cel6 CBHs are inactivated at lower temperatures than Cel7 CBHs, and this represents a limiting factor for industrial utilization. In this study, we produced a series of mutants of the glycoside hydrolase family 6 cellobiohydrolase Pc Cel6A from the fungus Phanerochaete chrysosporium , and compared their thermal stability. Eight mutants from a random mutagenesis library and one rationally designed mutant were selected as candidate thermostable mutants and produced by heterologous expression in the yeast Pichia pastoris . Comparison of the hydrolytic activities at 50 and 60 °C indicated that the thermal stability of Pc Cel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.     

Trichoderma enzymes promote Fibrobacter succinogenes S85 adhesion to,and degradation of,complex substrates but not pure cellulose

The effects of an enzyme preparation from Trichoderma longibrachiatum (TE) on adhesion and growth of the fibrolytic rumen bacterium Fibrobacter succinogenes S85 was studied to gain a better understanding of the action of feed enzyme additives on fibre digestion by ruminants. Adhesion experiments were performed on crystalline cellulose, corn silage and alfalfa hay. Adhesion of F succinogenes to cellulose was negatively related to the concentration of TE (p < 0.05). At the highest concentration used, TE reduced adhesion to cellulose from 65 to 39%. For corn silage and alfalfa hay, TE stimulated adhesion at low levels (p < 0.05) but this effect was lost at higher levels. Culture experiments were performed on crystalline cellulose and corn silage. The presence of TE in media containing cellulose failed to increase substrate disappearance or gas production although it increased numbers of non‐adherent bacteria (p < 0.05). When corn silage was used, the addition of TE increased NDF disappearance (p < 0.05) at 24 and 48 h (33 and 52% in controls versus 53 and 65% in TE treatments). Growth rate and gas production were also stimulated (p < 0.05). We conclude that, for cellulose, the hydrolytic enzymes in TE obstructed available binding sites decreasing bacterial adherence. Fibrobacter succinogenes digested cellulose efficiently and addition of exogenous cellulases did not further increase substrate disappearance. However, for complex plant substrates, low concentration of TE increased bacterial adhesion and plant (corn) fiber degradation. For the Department of Agriculture and Agri‐Food, Government of Canada, © Minister of Public Works and Government Services Canada 2004. Published for SCI by John Wiley & Sons, Ltd.     

Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose

The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16–2.5 mM glucose, with a correlation coefficient, r, of 0.998.     

Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis

Cyclodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. B1001 catalyzed the production predominantly of alpha-cyclodextrin (CD) from starch (Tachibana, Y. et al., Appl. Environ. Microbiol., 65, 1991-1997, 1999). The CGTase gene (cgtA) from this strain was cloned and sequenced. It was composed of 2217 nucleotides, and encoded a protein (739 amino acids) with a molecular mass of 83,240 Da. Recombinant CgtA expressed in Escherichia coli also catalyzed the production predominantly of alpha-CD from starch, as did native CgtA from strain B1001. Based on a substrate binding model of Bacillus circulans no. 8 CGTase, Tyr100, Trp191 and Tyr267 were specified to locate the spiral amylose and to minimize the size of the CD by saccharide aromatics interaction. In order to determine the critical residue for catalyzing production predominantly of alpha-CD, site-directed mutations were introduced in CgtA (Y100W, Tyr100-->Trp; W191Y, Trp191-->Tyr; W191F, Trp191-->Phe; Y267W, Tyr267-->Trp; Y267F, Tyr267-->Phe). Analysis of the reaction products by HPLC revealed that the mutant enzyme Y267W produced more beta- and gamma-CD than the wild-type enzyme. However, the other mutants still produced high levels of alpha-CD, suggesting that Tyr267 plays a critical role in alpha-CD production catalyzed by B1001 CGTase.     

Electron Spin Resonance Spectroscopy for the Identification of Irradiated Foods with Complex ESR Signals

Electron spin resonance (ESR) spectroscopy for the detection of irradiated food could provide complex spectra due to the presence of different nonirradiation-specific paramagnetic species in the sample. Particularly, ESR signals from naturally present Mn²⁺ could limit the ESR-based detection of irradiated foods of plant origin. In this study, the effects of different concentrations of Mn²⁺ on the radiation-specific ESR spectral features of radiation-induced crystalline sugar or cellulose radicals were examined. Soy sauce powder with Mn²⁺ was irradiated at 10 kGy and its effect on the identification of radiation-induced sugar (pak choi, 10 kGy) and cellulose (red pepper powder, 10 kGy) radicals was evaluated. In the blends of red pepper and soy sauce, the left (g ₁?=?2.025) and central (g ₂?=?2.005) peaks of the cellulose signal significantly decreased with the decline of red pepper content (R ²?=?0.9384, g ₁; 0.8819, g ₂). However, the right peak (g ₃?=?1.988) significantly increased (R ²?=?0.9353, g ₃) with an escalation in the soy sauce content. The cellulose radical signals were identifiable from the blends containing more than 50 % red pepper powder. In the blends of soy sauce and pak choi, the ESR intensity of crystalline sugar radicals significantly decreased with the decline in pak choi content (R ²?=?0.9887). However, Mn²⁺ signal didn’t overlap with the sugar radical signals. The ESR signals from radiation-induced crystalline sugar remained unaffected; however, the signals from cellulose radicals provided a reduced detectionability depending upon the concentration of Mn²⁺ in the sample.     

The binding mechanism of lecithin to soybean 11S and 7S globulins using fluorescence spectroscopy

Interactions between lecithin (Lec) and soybean 11S and 7S globulins were investigated using fluorescence spectroscopy. Fluorescence of 11S and 7S was quenched by Lec. The quenching mechanism was static due to formation of a complex. Thermodynamic parameters were measured using the van’t Hoff equation. Interactions of Lec with both globulins were driven by hydrophobic forces and binding processes were spontaneous. A larger absolute value of ΔG for 11S indicated that binding of 11S was much easier with Lec. Three-dimensional fluorescence spectra showed that binding of Lec with 11S more easily induced changes in the microenvironments of Trp and Tyr and in the polypeptide backbone structure of 11S. Simulated experiments verified that soybean proteins with a high 11S content combine both more easily and more strongly with Lec.     

Short communication: Roles of outer membrane protein W on survival,cellular morphology,and biofilm formation of Cronobacter sakazakii in response to oxidative stress

Cronobacter species are a group of opportunistic food-borne pathogens that cause rare but severe infections in neonates. Tolerance to environmental stress in Cronobacter is known; however, factors involved in oxidative stress are undefined. In this study, Cronobacter sakazakii survival, cellular morphology, and biofilm formation in response to oxidative stress were evaluated between the wild type (WT) and an outer membrane protein W (OmpW) mutant. The survival rates of ΔOmpW strain after treatment with 1.0 and 1.5 mM hydrogen peroxide were significantly reduced compared with those of WT. Morphological changes, including cell membrane damage and cell fragmentation, in ΔOmpW were more predominant than those in WT. By crystal violet staining, we also observed increased biomass in ΔOmpW biofilms as compared with WT following treatment with 0.5 and 1.0 mM H₂O₂. Biofilms using scanning electron microscopy and confocal laser scanning microscopy further confirmed the structural changes of biofilms between WT and ΔOmpW in response to oxidative stress. The current findings show that OmpW contributed to survival of planktonic cells under oxidative stress and the deletion of OmpW facilitated the biofilm formation in C. sakazakii to adapt to oxidative stress.     

Degradation of carrot (Daucus carota) fibres with cell-wall polysaccharide-degrading enzymes

Carrot Daucus carota L fibres were degraded with two enzyme preparations, SP249 from Aspergillus aculeatus and Celluclast from Trichoderma reesei. The enzymic activities of these complexes indicate that SP249 was particularly active on pectic polymers, and Celluclast could degrade amorphous and crystalline cellulose. A combination of both preparations degraded carrot fibres with a synergistic effect and led to the solubilisation of 95% of the cell-wall polysaccharides. The kinetics of solubilisation of sugars and gel-permeation chromatography of the soluble products show that pectic polymers were rapidly solubilised and then, in a second stage, degraded mainly to monomers, whereas cellulose was more slowly hydrolysed to cellobiose and glucose. Part (67%) of the polysaccharides were saccharified, the residual soluble material being rhamnogalacturonans containing arabinose residues. Residual insoluble fibres (10% of initial weight) were not liquefied and were composed mainly of lignin, proteins and polymers of glucose, xylose and galacturonic acid.     

Introduction of sulfhydryl groups and disulfide linkage to mungbean 8Sα globulin and effects on physicochemical and functional properties

Sulfhydryl groups and disulfide bond were introduced in mungbean's major storage protein, 8Sα globulin, by protein engineering to improve structural stability and functional properties. Five modified proteins or mutants (F59C, I99C, A213C, one free sulfhydryl group; I99C/A213, one disulfide bridge; F59C/I99C/A213C, one free sulfhydryl group and one disulfide linkage) were expressed in Escherichia coli at a yield similar to that of the unmodified protein or wild type (WT) in soluble form (38%). The number of introduced groups in the mutants was confirmed by Ellman analysis. Mutant and WT proteins exhibited similar elution patterns on gel filtration indicating their trimeric native conformation. Mutants had 2 to 3.8 °C higher T_m values than WT and were digested by chymotrypsin at 52–58% in 60 min but exhibited different digestion patterns. All mutants showed greater hardness of heat-induced gels than WT, especially I99C/A213C and F59C/I99C/A213C. Results indicate the improved structural stability of the modified 8Sα globulin.     

Studies on the Relationship Between Water-satured State and Crystallinity by the Diffraction Method for Moistened Potato Starch

Crystallinity was measured by X-ray diffraction method and water-saturated state was determined by means of the moisture contents calculated from the crystallinity for moistened cellulose and potato starch. In the X-ray diffractograms the upper areas which were separated by the straight line joined the points of intensity at 37° and 4° (8°) of diffraction angle was the relative reflection area in the crystalline and amorphous portions, and the lower area was the background of the non-relative reflection area. The upper area separated by the smooth curve joined the minimum intensity at 4° to 37° of diffraction angle corresponded to the crystalline portion and the lower area to the amorphous one. The ratio of the crystalline portion to sum of the crystalline and amorphous portions was considered to be crystallinity. Crystallinity of the air-dried and the moisture-saturated (28%) samples of cellulose were 0.68 and 0.71, resp. The crystallinity of the air-dried and water-saturated (49%) samples of potato starch were 0.24 and 0.32, resp.     

纤维素酶酶解苇浆纤维微观结构和结晶结构的变化

采用红外光谱(IR)、X射线衍射分析和扫描电镜对在纤维素酶解进程中苇浆纤维素大分子的结构及纤维形态的变化进行了研究.研究结果表明,纤维素酶酶解进程中,纤维素大分子的晶型没有改变,但结晶度呈现周期性变化,微晶尺寸略有降低.在酶解初期,纤维素酶不仅作用于纤维素无定形区,也开始作用在纤维素结晶区表面;在酶解中、后期,纤维素酶对纤维素结晶区和无定形区的作用呈现周期性变化.纤维素酶解过程中纤维表面呈现周期性"剥皮"现象,并且在纤维表面出现孔洞和沟槽,在孔洞处纤维易断裂,导致纤维长度和粗度的降低.     

Modeling of lactose crystallization and color changes in model infant foods

Lactose crystallization and color changes in formulas containing β-lactoglobulin and gelatinized starch were investigated. Model infant formulas were prepared by colyophilization of 3 components (lactose, β-lactoglobulin, and gelatinized starch). A mixture design was used to choose the percentage of each mixture component. These formulas were stored for 3 mo at different relative humidities (RH), ranging from approximately 0 to 94.6%, to study the lactose crystallization and color changes. Crystallization kinetics was studied by gravimetric methods, and lactose state (crystalline vs. amorphous) was verified before and after storage by differential scanning calorimetry. Before storage, lyophilized lactose was amorphous, but during storage it crystallized, depending on the RH. The lactose crystallization RH depended on the quantity of β-lactoglobulin and gelatinized starch, and by increasing these quantities, the crystallization RH increased. For some formulas, the crystallization RH was noted at 3 different RH during storage. The first was noted after 1 d of storage and the second and third were observed later on, showing that crystallization is a time-dependent phenomenon. Nonenzymatic browning was studied in model infant formulas by yellow color changes of samples at 11.3, 43.2, 54.5, and 75.4% RH. In this study, 7 mathematical models were proposed to predict the moisture sorption properties and color changes at different RH, and the models were validated by experimental results.     

Chemical and in vivo evaluation of a brown midrib mutant of Zea mays. II. Nuclear magnetic resonance spectra of digested and undigested alkali lignins and undigested dimethyformamide lignins

Nuclear magnetic resonance (n.m.r.) spectra were obtained on the alkali lignins extracted from Tr and bm₁ cornstalks, before and after digestion by sheep, and on the dimethylformamide lignins (DMFL) from undigested cornstalks. Differences due to solvent extraction, plant genotype and digestion were apparent. The bm¹ corn lignin has a higher degree of cross linking of the propane side chain of the lignin molecule than Tr corn lignin, a factor which may be significant in determining the effect of lignin on digestibility. The main effect of digestion was to increase the degree of cross-linking of the propane side chain, especially with the Tr lignin. Alkali lignin n.m.r. spectra were considerably different from DMFL n.m.r. spectra. For example over 40% of the total bm DMFL protons were highly shielded protons but these were virtually absent in the alkali lignins. These highly shielded protons cannot be explained by the presence of ferulic and p-coumaric acids in DMFL.     

金属-有机框架/纤维素复合材料研究进展

金属-有机框架（MOFs）是由金属离子和有机配体构成的具有一维、二维、三维结构的多功能晶态材料,MOFs本身的晶型结构决定了它们大多以粉末状存在,在回收、加工成型等方面受到限制。选择天然的或改性的柔性纤维素基材作为沉积MOFs的理想基底,一方面MOFs可赋予纤维素基材许多新的功能,实现纤维素的高值化利用;另一方面纤维素基材可为MOFs提供更广阔的利用空间,实现MOFs的多级开发利用。本文综述了MOFs/纤维素复合材料的研究进展,包括基底材料的选择、制备方法、性能及应用等。     

Ionized calcium requirement of rumen cellulolytic bacteria

Ionized calcium (Ca⁺²) appears to be required by the 3 predominant species of rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. The present study evaluated the role of ionized calcium in growth and cellulose digestion. Maximum growth or rate and extent of digestion and lag time were the criteria used to evaluate Ca⁺² requirements. All cultures except F. succinogenes A3c grew when repeatedly transferred in a medium without added Ca⁺². As Ca⁺² concentration increased in cellobiose medium, the rate of growth increased and lag time decreased for F. succinogenes A3c, whereas F. succinogenes S85 exhibited increases in both maximum growth and rate of growth. No responses in any of the criteria were observed for the ruminococci in cellobiose medium. Both strains of F. succinogenes had an absolute requirement for Ca⁺² with cellulose as the only substrate. For strain A3c the requirement was 0.36 to 0.42 mM and for S85, >0.64 mM. Increases in extent of cellulose degradation occurred with all strains of ruminococci as Ca⁺² concentration increased; however, degradation in Ca⁺²-free medium was similar to that of F. succinogenes with Ca⁺². Although the ruminococci presumably have cellulosomes that require Ca⁺² in their structure, such was not evident in our studies. The function of Ca⁺² in cellulose degradation by F. succinogenes is unknown, but may be related to the secretion or activation of their cellulolytic enzymes. Based on reported concentrations of Ca⁺² in the rumen, it seems unlikely that an in vivo deficiency would occur for these bacteria.     

Structural characterizations of waxy maize starch residue following in vitro pancreatin and amyloglucosidase synergistic hydrolysis

The objective of this work was to determine if in vitro digestion altered the molecular structure and slow digestion property of waxy maize starch. The Englyst testing on partially hydrolysis residual starches showed an increase of RDS accompanied a reduction of RS with increasing time of digestion, while SDS was almost constant. Scanning electron micrographs showed that the pattern of enzymatic hydrolysis was inside-out layer-by-layer digestion. A threefold decrease in the average molecular weight of starch components was observed after α-amylolysis for 120 min. There were increases in the onset temperature, peak temperature and ratio of absorbance 1047/1022 cm⁻¹, while the enthalpy of gelatinization, crystal structure, and crystallinity invaried. These changes suggest simultaneously enzymatic hydrolysis of both crystalline and amorphous regions of starch granule during in vitro digestion. SDS of starch residue also may consists of layered structure of amorphous and crystallite regions and located periphery of starch granules.     

Morphological,crystalline, thermal and physicochemical properties of cellulose nanocrystals obtained from sweet potato residue

Sweet potato residue (SPR), a by-product of sweet potato starch industry, was used as starting material to prepare cellulose nanocrystals. The method of sulfuric acid hydrolysis accompanied with ultrasonication and homogenization was used to prepare cellulose nanocrystals. The morphological, crystalline, thermal, and physicochemical properties of the cellulose nanocrystals were studied. SEM and TEM images showed spherical or elliptic granules of cellulose nanocrystals with sizes ranged from 20 to 40 nm. XRD analysis indicated that the cellulose nanocrystals retained the cellulose I crystalline structure, with a crystallinity of approximately 72.53%. TGA curves showed that the decomposition temperature of nanocrystals was decreased. These results showed that cellulose nanocrystals were successfully obtained from SPR and might be potentially applied in various fields, such as pharmaceutical and food additives, bio-nanocomposites, packaging, etc.     

INFLUENCE OF G187W/K188F/D189Y MUTATION IN THE SUBSTRATE BINDING POCKET OF TRYPSIN ON β-CASEIN PROCESSING

Undertaking to modulate the catalytic properties of trypsin, highly conserved G187, K188 and D189 were replaced with aromatic amino acid residues in order to perturb the electrostatics and to amplify hydrophobic interactions of the substrate binding site. The kinetic parameters of the wild-type trypsin and G187W/K188F/D189Y mutant were determined with synthetic tetrapeptide substrates and β-casein at different pHs. Compared with trypsin, the mutant G187W/K188F/D189Y exhibits 1.3-fold increase in K_m values for the substrates containing arginine and lysine. This mutant shows a 30- to 40-fold decrease of its kcat and its second-order rate constant k_cm/k_m decreases = 40- and 55-fold for substrates containing arginine and lysine, respectively. The kinetic analysis reveals that the mutant conserves the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. Surprisingly, as demonstrated only by proteolysis of a natural substrate (β-casein), the mutant cleaves also peptide bonds involving asparagine and glutamine.     

Subsite Structure of Rhizopus niveus Glucoamylase,Estimated with the Binding Parameters for Maltooligosaccharides

Based on K_d and K_i for maltosaccarides G_n (n = 1 ± 7) and on a specified mode of their binding, the intrinsic affinities A₁, A₂, –, A_i at subsites i (i = 1 ± 5), were tried to estimate for glucoamylase from Rhizopus niveus. Previously, we have evaluated the apparent values A_i using K_m and k₀ on the enzyme-catalyzed hydrolysis for G_n, where A₁ is nearly 0 kcal/mol. Thus A₁, which was found here 3.3 kcal/mol, may be cancelled out with the heat, which is consumed for the hydrolytic cleavage of a substrate glucosyl bond.