4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Efficiency of lignin biosynthesis: a quantitative analysis

Lignin is derived mainly from three alcohol monomers: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Biochemical reactions probably responsible for synthesizing these three monomers from sucrose, and then polymerizing the monomers into lignin, were analysed to estimate the amount of sucrose required to produce a unit of lignin. Included in the calculations were amounts of respiration required to provide NADPH (from NADP(+)) and ATP (from ADP) for lignin biosynthesis. Two pathways in the middle stage of monomer biosynthesis were considered: one via tyrosine (found in monocots) and the other via phenylalanine (found in all plants). If lignin biosynthesis proceeds with high efficiency via tyrosine, 76.9, 70.4 and 64.3 % of the carbon in sucrose can be retained in the fraction of lignin derived from p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, respectively. The corresponding carbon retention values for lignin biosynthesis via phenylalanine are less, at 73.2, 65.7 and 60.7 %, respectively. Energy (i.e. heat of combustion) retention during lignin biosynthesis via tyrosine could be as high as 81.6, 74.5 and 67.8 % for lignin derived from p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, respectively, with the corresponding potential energy retention values for lignin biosynthesis via phenylalanine being less, at 77.7, 69.5 and 63.9 %, respectively. Whether maximum efficiency occurs in situ is unclear, but these values are targets that can be considered in: (1) plant breeding programmes aimed at maximizing carbon or energy retention from photosynthate; (2) analyses of (minimum) metabolic costs of responding to environmental change or pest attack involving increased lignin biosynthesis; (3) understanding costs of lignification in older tissues; and (4) interpreting carbon balance measurements of organs and plants with large lignin concentrations.     

The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism

The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants; however, no conditions suitable for the unambiguous assay of the enzyme are known. As a result, all attempts to purify the protein and clone its corresponding gene have failed. By screening for plants that accumulate reduced levels of soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. Using radiotracer-feeding experiments, we have determined that the ref8 mutant is unable to synthesize caffeic acid, suggesting that the mutant is defective in a gene required for the activity or expression of C3H. We have isolated the REF8 gene using positional cloning methods, and have verified that it encodes C3H by expression of the wild-type gene in yeast. Although many previous reports in the literature have suggested that C3H is a phenolase, the isolation of the REF8 gene demonstrates that the enzyme is actually a cytochrome P450-dependent monooxygenase. Although the enzyme accepts p-coumarate as a substrate, it also exhibits significant activity towards other p-hydroxylated substrates. These data may explain the previous difficulties in identifying C3H activity in plant extracts and they indicate that the currently accepted version of the lignin biosynthetic pathway is likely to be incorrect.     

Functional genomics of wood quality and properties

Genomics promises to enrich the investigations of biology and biochemistry. Current advancements in genomics have major implications for genetic improvement in animals, plants, and microorganisms, and for our understanding of cell growth, development, differentiation, and communication. Significant progress has been made in the understanding of plant genomics in recent years, and the area continues to     

Anti-u.v. activity of lignin biopolymers on Euglena gracilis

Sulphur-free lignin biopolymer and its oxidized and reduced derivatives have been prepared and their inhibitory activity against u.v.-induced mutagenesis in Euglena gracilis was evaluated. The structure- and dose-dependent anti-u.v. activity of lignins was observed at concentrations higher than 250gml^–1. The oxidized lignin showed the most antimutagenic activity, followed by the reduced lignin and the unmodified lignin had the least antimutagenic activity.     

Residual lignin studies of laccase-delignified kraft pulps

The delignification of chemical pulps with laccase and -hydroxybenzotriazole was explored employing a pre- and post-O₂ delignified softwood draft pulp. The delignification properties of laccase were shown to be improved with -hydroxybenzotriazole was used as a mediator instead of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Analysis of the structure of residual lignin before and after laccase/ -hydroxybenzotriazole treatment indicated that the biobleaching system oxidizes the phenolic component of lignin and that the residual lignin is demethylated and significantly enriched in carboxylic acid groups.     

Lignans of Larix leptolepis

Five lignans have been isolated from wood of Larix leptolepis. They are identified as 1-(4-hydroxy-3-methoxyphenyl)-2-4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy-propane- 1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-2-methoxy-4-[1-(E)-propen-3-ol]-phenoxy- propane-1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-(4-formyl-2-methoxyphenoxy)-propane-1,3-diol, 1,2-bis-(4-hydroxy-3-methoxyphenyl)-propane-1,3-diol and a trilignol, leptolepisol C.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.     

不同地下水位灰化薹草分解过程中木质素降解与碳、氮、磷释放的关系

为研究湿地植物分解过程中木质素分解与碳、氮、磷元素释放之间的关系及其对不同地下水位环境梯度的响应,在鄱阳湖典型碟形湖泊-白沙湖设置了200 m×300 m分解试验样地,沿地下水位梯度划分4个试验样带(从湖岸到湖心依次为GT-A, GT-B, GT-C, GT-D),采用分解袋法模拟了典型湿地植物灰化薹草在不同地下水位环境的分解过程。结果表明,分解15 d后,地下水位的升高促进了木质素的分解和碳、氮、磷元素的释放;在分解第60—90天,碳、氮、磷元素的相对归还指数随着木质素分解速率的升高而显著增大,并且相关性程度随着分解时间先增强,后减弱;分解过程中土壤pH、含水量和微生物量碳、氮对木质素的分解和碳、氮、磷元素的归还具有显著的促进作用。研究结果将有助于深化对湿地植物分解和生物地球化学循环过程的认识,为湿地自然保护区的科学有效管理提供理论依据。