Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis: Part II

Lignocellulose breakdown in biorefineries is facilitated by enzymes and physical forces. Enzymes degrade and solubilize accessible lignocellulosic polymers, primarily on fiber surfaces, and make fibers physically weaker. Meanwhile physical forces acting during mechanical agitation induce tearing and cause rupture and attrition of the fibers, leading to liquefaction, that is, a less viscous hydrolysate that can be further processed in industrial settings. This study aims at understanding how mechanical agitation during enzymatic saccharification can be used to promote fiber attrition. The effects of reaction conditions, such as substrate and enzyme concentration on fiber attrition rate and hydrolysis yield were investigated. To gain insight into the fiber attrition mechanism, enzymatic hydrolysis was compared to hydrolysis by use of hydrochloric acid. Results show that fiber attrition depends on several factors concerning reactor design and operation including drum diameter, rotational speed, mixing schedule, and concentrations of fibers and enzymes. Surprisingly, different fiber attrition patterns during enzymatic and acid hydrolysis were found for similar mixing schedules. Specifically, for tumbling mixing, slow continuous mixing appears to function better than faster, intermittent mixing even for the same total number of drum revolutions. The findings indicate that reactor design and operation as well as hydrolysis conditions are key to process optimization and that detailed insights are needed to obtain fast liquefaction without sacrificing saccharification yields.     

Determination of the number-average degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis

A rapid and accurate method for determining the number-average degree of polymerization (DP(n)) was established for insoluble cellulose and soluble cellodextrins as the ratio of glucosyl monomer concentration determined by the phenol-sulfuric acid method divided by the reducing-end concentration determined by a modified 2,2'-bicinchoninate (BCA) method. The modified BCA method, featuring incubation at 75 degrees C for 30 min, did not result in beta-glucosidic bond cleavage, whereas substantial cleavage was observed at higher temperature. Solubilization of insoluble cellulose in cold phosphoric acid prior to measurement of the reducing-end concentration by the BCA method was found not to be necessary for several model celluloses such as microcrystalline cellulose, but such solubilization was required for large fibers of cellulose such as Whatman No. 1 filter paper. The phenol-sulfuric acid method can be used for measuring the glucosyl monomer concentration of soluble cellodextrins, and also for insoluble cellulose if preceded by a liquefaction step. Standard deviations of < or =2% were obtained for both reducing and glucosyl monomer determination and of < or =3% for overall determination of DP. By use of the reported method, hydrolysis of phosphoric acid-swollen cellulose (PASC) by the Trichoderma reesei cellulase system was shown to result in a rapid decrease in DP as hydrolysis proceeded. By contrast, the DP of Avicel remained nearly constant during hydrolysis. The specific enzymatic cellulose hydrolysis rate is 100-fold higher for PASC as compared to Avicel.     

Liquefaction of lignin by polyethyleneglycol and glycerol

Enzymatic hydrolysis lignin (EHL), isolated from the enzymatic hydrolysis residues of the biomass, was liquefied using the mixed solvents of polyethyleneglycol (PEG) and glycerol at the temperature of 130-170°C with sulfuric acid as a catalyst. The influences of liquefaction parameters, such as the molecular weight of PEG, mass ratio of sulfuric acid to EHL, liquefaction temperature and time, and mass ratio of liquid (liquefying cosolvent) to solid (EHL) on the residue content and hydroxyl number were discussed. The FT-IR spectrum result showed that the liquefaction product of EHL was polyether polyol. The hydroxyl number of the liquefaction product was 80-120 mgKOH/g higher than that of PEG.     

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.     

SO3H-functionalized ionic liquid: efficient catalyst for bagasse liquefaction

Liquefaction is a process for the production of biofuel or value-added biochemicals from non-food biomass. SO₃H-, COOH-functionalized and HSO₄-paired imidazolium ionic liquids were shown to be efficient catalysts for bagasse liquefaction in hot compressed water. Using SO₃H-functionalized ionic liquid, 96.1% of bagasse was liquefied and 50.6% was selectively converted to low-boiling biochemicals at 543 K. The degree of liquefaction and selectivity for low-boiling products increased and the average molecular weight of the tetrahydrofuran soluble products decreased with increasing acidic strength of ionic liquids. Analysis of products and comparative characterization of raw materials and residues suggested that both catalytic liquefaction and hydrolysis processes contribute to the high conversion of bagasse. A possible liquefaction mechanism based on the generation of 3-cyclohexyl-1-propanol, one of the main products, is proposed.     

响应面法优化米糠淀粉酶法水解工艺

以米糠为原料,对米糠淀粉酶法水解生产葡萄糖的液化工艺进行研究和优化,来提高葡萄糖收率。在单因素试验的基础上,用响应面法对液化工艺进行优化。结果表明,液化工艺的最佳条件为酶用量0.11%、醪浓度25%、pH=6.0、温度88℃,在此条件下得到的液化葡萄糖值(即DX值)平均值为6.54%。然后对此液化液进行糖化,最终得到的糖化液DX值为97.07%。     

Stereospecific hydrolysis by soluble and immobilized lipases

Stereospecific hydrolysis of insoluble monoesters by lipases are reported. Among the lipases tested, porcine pancreatic lipase was the most stereospecific when acting on 3-chloro-2-methyl propanol propionate. When the chain length of the acid was enhanced, the stereospecificity decreased. Initial rate measurements analysis concluded that the observed stereospecificity was the result of different catalytic constants rather than different Michaelis constants. From these results, methods were derived for the preparation of l- or d-3-chloro-2-methylpropanol (an intermediary in the synthesis of levomepromasine) based on the hydrolysis of esters by soluble or immobilized lipases.     

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Linkage between the teichuronic acid and mucopeptide components

1. After extraction of teichoic acid from cell walls of Bacillus licheniformis with dilute alkali, the insoluble residue contains the teichuronic acid and mucopeptide components and a small amount of residual phosphorus. 2. A complex of teichuronic acid and a part of the mucopeptide was isolated from the soluble fraction obtained by lysozyme treatment of alkali extracted walls. 3. Small-molecular-weight mucopeptide fragments, not containing teichuronic acid, are obtained from the soluble fraction in yields similar to those obtained after treatment of whole walls or acid-extracted walls with lysozyme. 4. The covalent linkages between teichuronic acid and mucopeptide are broken by treatment with dilute acid. The release of teichuronic acid chains is accompanied by the hydrolysis of N-acetylgalactosaminide linkages and the exposed N-acetylgalactosamine residues form chromogen under very mild conditions, indicating that they are substituted on C-3. 5. The initial rate of formation of reactive N-acetylgalactosamine residues during mild acid hydrolysis is parallel to the rate of extraction under the same conditions of teichuronic acid from alkali-treated insoluble walls, and to the rate of acid hydrolysis of glucose 1-phosphate. 6. The results suggest that the teichuronic acid chains are attached through reducing terminals of N-acetylgalactosamine residues to phosphate groups in the mucopeptide. 7. Muramic acid phosphate was isolated from the insoluble mucopeptide remaining after extraction of walls with dilute alkali followed by dilute acid.     

Evidence that pancreatic lipase is responsible for the hydrolysis of cutin,a biopolyester present in mammalian diet,and the role of bile salt and colipase in this hydrolysis

The enzyme, which catalyzes hydrolysis of cutin, an insoluble biopolyester of hydroxy and epoxy fatty acids, was purified from porcine pancreas. With three different purification methods, previously used for the purification of pancreatic lipase, it is shown that cutin hydrolase is pancreatic lipase. This enzyme released oligomers and all types of monomers from the polymer with a pH optimum around 7.5. Taurodeoxycholate inhibited cutin hydrolysis by lipase and colipase reversed this inhibition. Evidence is presented which suggests that bile salt stabilizes the enzyme at the surface of the insoluble substrate and that the interaction of the polymer surface with the lipase-colipase-bile salt system is similar to that previously observed with triglycerides. Diethyl-p-nitrophenyl phosphate inhibited cutin hydrolysis by lipase but the hydrolysis was insensitive to diisopropyl fluorophosphate.     

Separation and characterization of the soluble and insoluble components of insoluble laminaran

Insoluble laminaran, a (1→3)-β-D-glucan from Laminaria hyperborea (L. cloustoni), has been fractionated by differential solubility into soluble and insoluble fractions. These fractions were degraded with a purified exo-(1→3)-β-D-glucanase from Basidiomycete sp. QM806 giving, as primary hydrolysis products, D-glucose, gentiobiose, laminarabiose, and 1-O-β-laminarabiosylmannitol. Gentiobiose was obtained in only trace amounts from the insoluble fraction of laminaran, suggesting the absence of branching. Successive application of periodate oxidation, reduction, mild acid hydrolysis, and enzymic degradation indicated that the branch in the soluble fraction consists of a single β-(1→6)-linked D-glucosyl residue. The results indicate that “insoluble” laminaran is apparently an aggregate of three closely related polysaccharide species: a soluble, branched, reducing component (soluble laminarose); an insoluble, unbranched, reducing component (insoluble laminarose); and an unbranched, nonreducing component (laminaritol) that has a monosubstituted mannitol residue at the reducing terminal. Laminaritol was found to be about equally distributed between the soluble and insoluble fractions. The average d.p. of the laminaran components is 20–25 residues, as determined from the relative amounts of enzymic hydrolysis products and from periodate-oxidation data.     

Starch-binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI > GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.     

Hydrolysis of nucleic acids by sepharose-micrococcal endonuclease

Initial reaction rates for the hydrolysis of nucleic acids with micrococcal endonuclease (EC 3.1.31.1) insolubilized on Sepharose are strongly influenced by diffusional limitations. Although the absolute values are low, they can be increased substantially by changing particle and pore size of the support, or enzyme concentration in the insoluble derivative. As a result of steric and diffusional limitations, the course of the reaction and selectivity to hydrolysis products for the insoluble derivatives are different to those of the native enzyme; the former produces mainly large and small fragments but few of intermediate size. Because of these differences in course and selectivity of the reaction, diffusional limitations become less important when high initial reaction rates are not required.     

Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels

A sensitive staining method for alkali-labile phosphoproteins has been developed. As little as 0.2 nmol bound P/mm2 can be detected. The procedure is based on alkaline hydrolysis, phosphate capture, and formation of an insoluble rhodamine B-phosphomolybdate complex. A further modification for the qualitative detection of alkaline phosphatase activity on polyacrylamide gels is proposed. During incubation, the released Pi is precipitated as lead phosphate and subsequently stained with rhodamine B.     

The C-terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis

The chitinase gene chi1 of Aeromonas caviae CB101 encodes an 865-amino-acid protein (with signal peptide) composed of four domains named from the N-terminal as an all-beta-sheet domain ChiN, a triosephosphate isomerase (TIM) catalytic domain, a function-unknown A region, and a putative chitin-binding domain (ChBD) composed of two repeated sequences. The N-terminal 563-amino-acid segment of Chi1 (Chi1DeltaADeltaChBD) shares 74% identity with ChiA of Serratia marcescens. By the homology modeling method, the three-dimensional (3D) structure of Chi1DeltaADeltaChBD was constructed. It fit the structure of ChiA very well. To understand fully the function of the C-terminal module of Chi1 (from 564 to 865 amino acids), two different C-terminal truncates, Chi1DeltaChBD and Chi1DeltaADeltaChBD, were constructed, based on polymerase chain reaction (PCR). Comparison studies of the substrate binding, hydrolysis capacity, and specificity among Chi1 and its two truncates showed that the C-terminal putative ChBD contributed to the insoluble substrate-protein binding and hydrolysis; the A region did not have any function in the insoluble substrate-protein binding, but it did have a role in the chitin hydrolysis: Deletion of the A region caused the enzyme to lose 30-40% of its activity toward amorphous colloidal chitin and soluble chitin, and around 50% toward p-nitrophenyl (pNP)-chitobiose pNP-chitotriose, and its activity toward low-molecular-weight chitooligomers (GlcNAc)3-6 also dropped, as shown by analysis of its digestion processes. This is the first clear demonstration that a domain or segment without a function in insoluble substrate-chitinase binding has a role in the digestion of a broad range of chitin substrates, including low-molecular-weight chitin oligomers. The reaction mode of Chi1 is also described and discussed.     

Immobilization of a thermostable α‐amylase by covalent binding to an alginate matrix increases high temperature usability

Thermostable α‐amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95°C. 1‐ethyl‐3‐(3‐dimethylamino‐propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized α‐amylase were investigated. Covalently bound thermostable α‐amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95°C in a stirred batch reactor. The free and covalently bound α‐amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis‐Menten constant (K_m) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V_max) of immobilized and free enzyme were determined to be 10.4‐mg starch degraded/mL min mg bound protein and 25.7‐mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable α‐amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

13C-n.m.r. study of C hordein.

Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan.     

Optimization of the preparation of aqueous suspensions of waxy maize starch nanocrystals using a response surface methodology

Response surface methodology was used to investigate the effect of five selected factors on the selective H(2)SO(4) hydrolysis of waxy maize starch granules. These predictors were temperature, acid concentration, starch concentration, hydrolysis duration, and stirring speed. The goal of this study was to optimize the preparation of aqueous suspensions of starch nanocrystals, i.e., to determine the operative conditions leading to the smallest size of insoluble hydrolyzed residue within the shortest time and with the highest yield. Therefore empirical models were elaborated for the hydrolysis yield and the size of the insoluble residues using a central composite face design involving 31 trials. They allowed us to show that it was possible to obtain starch nanocrystals after only 5 days of H(2)SO(4) hydrolysis with a yield of 15 wt % and having the same shape as those obtained from the classical procedure after 40 days of HCl treatment, with a yield of 0.5 wt %.     

Properties of a reversible soluble-insoluble cellulase and its application to repeated hydrolysis of crystalline cellulose

Cellulase was covalently immobilized on an enteric coating polymer, Eudragit L, that is reversibly soluble and insoluble depending on the pH of the medium. The hydrolysis of solid cellulose with the immobilized enzyme can take advantage of the soluble property of the immobilized enzyme itself at the most reactive pH value; on the other hand, recovery of the enzyme can take advantage of the insoluble property of the enzyme at other pH values. It was experimentally confirmed that 100% of immobilized enzyme activity in solution can be recovered by precipitation and by dissolving it again by alternative change of pH. After a period of hydrolysis, immobilized enzyme and unreacted cellulose were precipitated together to remove the product-the soluble sugar solution-by changing pH. Following this, a new buffer solution was added to the precipitate to dissolve it and resume the reaction. This was repeated several times. The hydrolysis rate of this process increased significantly compared with that of a batch process. Utilization of the reversible soluble-insoluble carrier for immobilizing enzyme is promising, not only for cellulose-cellulase systems, but also for other heterogeneous reaction systems.     

A new assay for proteases using fluorescent labeling of proteins

A simple assay for proteases based on the fluorescent labeling of insoluble proteins (fibrin) or of soluble casein by 2-methoxy-2,4-diphenyl-3(2H)furanone has been developed. Fluorescence of the liberated peptide-fluorophors resulting from enzymatic hydrolysis is easily measured in the supernatant after separation of the unreacted fluorescent fibrin by centrifugation or from unreacted casein-fluorophor by acid precipitation. Nanogram quantities of trypsin, chymotrypsin, and elastase can be measured.