bImproving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) from Achaetomium sp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues-D244 and D299 -were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (T max ), temperature at which 50% of the maximal activity of an enzyme is retained (T 50 ), and melting temperature (T m ), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t 1/2 ) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ؎ 130 U/mg versus 28,000 ؎ 293 U/mg); on the other hand, it showed more residual activity (8,400 ؎ 83 U/mg versus 1,400 ؎ 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability. P ectin, the third most abundant polysaccharide of the plant cell wall, is the most structurally complex polysaccharide consisting of covalently linked galacturonic acid (1, 2). Pectinases-microbial enzymes which are capable of depolymerizing pectinplay an important role in wide biotechnological applications, including the fruit juice, vinification, paper, and textile industries, as well as the extraction of oils (3). Polygalacturonase (PG) is the most widely studied pectinase due to its high activity, mesophilic and acidic properties, and satisfactory performance in juice clarification, extraction, viscosity reduction, and yield improvement (4). Based on sequence similarity and action mode (5), PGs are classified into family 28 of glycoside hydrolase (GH) and endoand exo-types. Most endo-PGs are highly active and stable at 30 to 50°C (6; http://www.brenda-enzymes.org/), which are far from the requirements of thermophilic processing in the feed industry (7).To obtain a thermostable, highly active PG, either mining new genetic resources of thermophiles (8) or engineering the protein and application environment (9, 10) is the most popular practice. Thermophilic fungi Thielavia arenaria XZ7 (60°C) (7), Thermoascus aurantiacus CBMAI-756 (60 to 65°C) (11), Penicillium sp. strain SPC-F 20 (60°C) ...

Liriodendron chinense (Hemsl.), a Tertiary relic tree, is mainly distributed in subtropical China. The causes of the geographical distribution pattern of this species are poorly understood. In this study, we inferred historical dispersal routes and glacial refugia of this species by combining genetic data (chloroplast DNA (cpDNA), nuclear ribosomal DNA (nrDNA), and nuclear DNA (nDNA)) and geospatial data (climate and geology) with the methods of landscape genetics. Additionally, based on sequence variation at multiple loci, we employed GenGIS and Barrier software to analyze L. chinense population genetic structure. Dispersal corridors and historical gene flow between the eastern and western populations were detected, and they were located in mountainous regions. Based on species distribution model (SDMs), the distribution patterns in paleoclimatic periods were consistent with the current pattern, suggesting the presence of multiple refuges in multiple mountainous regions in China. The genetic structure analysis clustered most eastern populations into a clade separated from the western populations. Additionally, a genetic barrier was detected between the eastern and western populations. The dispersal corridors and historical gene flow detected here suggested that the mountains acted as a bridge, facilitating gene flow between the eastern and western populations. Due to Quaternary climatic fluctuations, the habitats and dispersal corridors were frequently inhabited by warm-temperate evergreen forests, which may have fragmented L. chinense habitats and exacerbated the differentiation of eastern and western populations. Ultimately, populations retreated to multiple isolated mountainous refugia, shaping the current geographical distribution pattern. These dispersal corridors and montane refugia suggested that the mountains in subtropical China play a crucial role in the conservation of genetic resources and migration of subspecies or related species in this region.

Taxodium 'zhongshansa', an extremely floodtolerant tree species, has been widely planted in eastern China due to its environmental attributes. Although much progress has been made in understanding its physiological response to flooding stress, as well as its rapid propagation and variety selection, little is known regarding its molecular genetics due primarily to a lack of reliable molecular markers. In this paper, 108,692 expressed sequence tags (ESTs) with a total size of 69.3 Mb derived from T. 'zhongshansa' were analyzed. And 10,038 simple sequence repeat (SSR) loci were identified from 8137 SSR-containing EST sequences. The average SSR frequency in the transcriptome was one in 6.90 kb of EST sequences. The most abundant repeat type was mononucleotide (6581, 65.56 %), followed by trinucleotide (2246, 22.37 %) and dinucleotide (1080, 10.76 %). After filtering unqualified SSR loci, loci with mononucleotide repeats and compound SSRs, 1958 EST-SSR loci were selected to design SSR makers. To validate this set of SSR markers, 503 primer pairs were randomly selected to amplify across 12 genomic DNA templates from three Taxodium species. Two hundred fifty-seven (51.09 %) out of 503 primer pairs amplified the expected products, of which 81 and 176 could amplify polymorphic and monomorphic bands, respectively. The functional categorization of EST sequences containing randomly selected markers revealed that 264 (52.49 %) had homology with known proteins. This set of EST-SSR markers will provide a valuable genetic and genomic tool for further genetic research in Taxodium, such as genetic map construction, quantitative trait loci mapping, and marker assisted selection.

Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered K m values from 0.32 (wild type PG8fn) to 0.75–4.74 mg/ml, all mutants displayed remarkably lowered k cat (~3–20,000 fold) and k cat/K m (~8–187,500 fold) values and significantly increased △(△G) values (5.92–33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.