Yeong-Suk Kim

Characterization of a recombinant β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus

A recombinant beta-glucosidase from Caldicellulosiruptor saccharolyticus DSM 8903 with a specific activity of 13 U/mg was purified by heat treatment and His-Trap affinity chromatography and identified as a single 54 kDa band on SDS-PAGE. The molecular mass of the native enzyme was 108 kDa as a dimer by gel filtration. beta-Glucosidase showed optimum activity at pH 5.5 and 70 degrees C for p-nitrophenyl (pNP)-beta-d-glucopyranoside. The half-lives of the enzyme at 60, 70, and 80 degrees C were 250, 24.3, and 0.4 h, respectively. The enzyme exhibited catalytic efficiency and specific activity for pNP-beta-d-fucopyranoside, pNP-beta-d-glucopyranoside, and pNP-beta-d-galactopyranoside in decreasing order among aryl-beta-glycosides, but not for aryl-alpha-glycosides. Cello-oligosaccharides from n = 2 to 5 as substrates using 4 mM each sugar and 3 U/mg of enzyme were completely hydrolyzed to glucose at 70 degrees C within 16 h.

Symbiotic adaptation of bacteria in the gut of Reticulitermes speratus: Low endo-β-1,4-glucanase activity

The termite is a good model of symbiosis between microbes and hosts and possesses an effective cellulose digestive system. Oxygen-tolerant bacteria, such as Dyella sp., Chryseobacterium sp., and Bacillus sp., were isolated from Reticulitermes speratus gut. Notably, the endo-beta-1,4-glucanase (EG) activity of all 16 strains of isolated bacteria was low. Due to the combined activity of EG from the termites and their symbiotic protozoa, the bacteria might not be compelled to express EG. This observation demonstrates how well intestinal bacteria have assimilated themselves into the efficient cellulose digestive systems of termites.

Rapid saccharification for production of cellulosic biofuels

The economical production of biofuels is hindered by the recalcitrance of lignocellulose to processing, causing high consumption of processing enzymes and impeding hydrolysis of pretreated lignocellulosic biomass. We determined the major rate-limiting factor in the hydrolysis of popping pre-treated rice straw (PPRS) by examining cellulase adsorption to lignin and cellulose, amorphogenesis of PPRS, and re-hydrolysis. Based on the results, equivalence between enzyme loading and the open structural area of cellulose was required to significantly increase productive adsorption of cellulase and to accelerate enzymatic saccharification of PPRS. Amorphogenesis of PPRS by phosphoric acid treatment to expand open structural area of the cellulose fibers resulted in twofold higher cellulase adsorption and increased the yield of the first re-hydrolysis step from 13% to 46%. The total yield from PPRS was increased to 84% after 3h. These results provide evidence that cellulose structure is one of major effects on the enzymatic hydrolysis.

Characterization of Cellulases from Schizophyllum commune for Hydrolysis of Cellulosic Biomass

The optimum culture condition of Schizophyllum commune for the cellulase production and its enzymatic characteristics for saccharification of cellulosic biomass were analyzed. S. commune secrets -1,4-xylosidase (BXL) and cellulases, including endo--1,4-glucanase (EG), cellobiohydrolase (CBH), and -glucosidase (BGL). The optimum reaction temperature for all cellulases was and the thermostable range was C. The optimum reaction pH for all cellulases was 5.5 in a range of temperature from to . The best nutritions for the cellulase production of S. commune among tested nutrients were 2% cellulose for the carbon source and corn steep liquor or peptone/yeast extract for the nitrogen source without vitamins. The environmental culture condition for the cellulase production was 5.5~6.0 for pH at . The enzyme activities of EG, BGL, CBH, and BXL were 3670.5, 631.9, 398.5, and 15.2 U/, respectively, after concentration forty times from the culture broth of S. commune which was grown at the optimized culture condition. Alternative filter paper unit assay showed 11 FPU/ enzyme activity. The saccharification tests using cellulase of S. commune showed the low saccharification rate on tested hardwoods but a high value of 50.5% on cellulose, respectively. The saccharification rate (50.5%) of cellulose by cellulase produced in this work is higher than 45.7% in the commercial enzyme (Celluclast 1.5L, 30 FPU/g, glucan).

Integrated analysis for die design including brittle damage evolution

Abstract Brittle failure mechanism has been well known as growth of penny-shaped micro-cracks, referred as brittle damage, which eventually cause macro-cracks and failure. A brittle damage model is proposed, which consists of a damage function and an evolution equation for brittle damage. The damage function determines whether a micro-crack begins to grow as a yield function in plasticity, while an evolution equation gives the growth rate of a micro-crack when it grows. An integrated analysis is then proposed to model the shrink-fit, elastic deformation, fatigue, wear as well as brittle damage evolution in a die. Applications of the proposed analysis to the extrusion die with multi-layers of stress-rings are given.

Current Research Trends in Wood Preservatives for Enhancing Durability - A Literature Review on Non-Copper Wood Preservatives -*

Current research trends of non-copper wood preservatives for enhancing durability was re- viewed; as a follow-up of the review in 2012 on copper-based wood preservatives. Main environ- mental friendly non-copper wood preservatives studied by many scientists were boron-based compounds, synthetic compounds from natural products, and pyrethroids family of chemicals, etc.

Complete Genome Sequence of Elizabethkingia sp. BM10, a Symbiotic Bacterium of the Wood-Feeding Termite Reticulitermes speratus KMT1

ABSTRACT Elizabethkingia sp. BM10 was isolated from the hindgut of the wood-feeding termite Reticulitermes speratus KMT1. It had cellobiohydrolase and β-glucosidase activities but not endo-β-glucanase activity. The complete sequence of its genome, which has a total size of 4,242,519 bases, is reported here. The genomic analysis identified six β-glucosidase candidate genes and three β-glucanase candidate genes.

A New α-Amylase from Reticulitermes speratus KMT1

ABSTRACT Termites are wood pests that cause vast economic damage every year. They digest both cellulose and starch, but the enzymes for starch digestion have not been well characterized. We obtained complete amino acid sequence information on the KME1 α-amylase from Reticulitermes speratus KMT1 through analysis of total mRNA sequences. The KME1enzyme has two α-amylase domains and is 68% identical to the α-amylase from Blattellager manica, its closest rela-tive in the GenBank database. Some unique features of its conserved region and its distant evolutionary relationship toother insect α-amylases suggest that KME1 is a new type of α-amylase.Keywords : Termite, Reticulitermes speratus KMT1, α-amylase, Homologous search, Phylogenetic analysis 1. INTRODUCTION 1) Termites efficiently degrade woody cellulose through a symbiotic collaboration with gut mi-croorganisms, and they absorb the degradation products as nutrients (Nakashima et al., 2002; Watanabe et al., 1997; Zhou et al., 2007). In the first step of cellulose degradation, termites secrete an endo-β-1,4-glucanase from their sali-vary glands that enzymatically reduces the size of cellulose. Later, symbiotic microorganisms in the termite gut secrete cellobiohydrolase and β-glucosidase to convert cellulose into glucose (Cho et al., 2010; Nakashima et al., 2002). Since starch is an energy-storing material in biological systems, termites should be able to use it as food. It has been shown that Coptotermes for-mosanus can survive on starch or glucose as a sole nutrient as well as on cellulose, and it does this without the assistance of symbiotic protozoans (Kanai et al., 2008). However, only the sequence of a partial catalytic domain of the amylase gene from Coptotermes formosanus has so far been reported (GenBank Accession number: KC740998); no complete sequence is available. Amylases are classified into three groups, α- amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), and γ-amylase (EC 3.2.1.3), depending on where they cleave the α(1 → 4) glucosidic linkage on

Effect of Carbon Source on the Hydrolytic Ability of the Enzyme from Fomitopsis pinicola for Lignocellulosic Biomass

In this study, effect of carbon source on the hydrolytic ability of the enzyme from Fomitopsis pinicola, a brown rot fungi, for lignocellulosic biomass were examined on two lignocellulosic biomasses (rice straw and wood) without any pretreatment. Cellulase activities of crude enzyme from F. pinicola, which was cultured on softwood mixture as a carbon source, were 19.10 U/ for endo--1,4-gulcanase (EG), 36.1 U/ for -glucosidase (BGL), 7.27 U/ for cellobiohydrolase (CBH), and 7.12 U/ for -1,4 xylosidase (BXL). Softwood mixture as a carbon source in F. pinicola comparatively enhanced cellulase activities than rice straw. The optimal pH and temperature of the cellulase was identified to pH 5 and for the hydrolysis of rice straw. Under these condition rice straw was hydrolyzed to glucose by the cellulase up to based on the glucan amount of the rice straw for 72 h, while the hydrolytic capability of commercial enzyme (Celluclast 1.5) from rice straw to glucose was estimated to at the same experimental condition. In case of addition of Tween 20 (0.1% w/w, substrate) to the cellulase the hydrolysis of rice straw to glucose was enhanced to .

Enzymatic Hydrolysis of Rice Straw, a Lignocellulosic Biomass, by Extracellular Enzymes from Fomitopsis palustris

ABSTRACT In the enzymatic hydrolysis of rice straw and wood meals using extra-cellular enzymes from Fomitopsis palustris, key factors which enhanced the sugar conversion yield were investigated in this work, such as enzyme production and enzyme reaction conditions, surfactant effects, and the surface structure of substrates. F. palustris cultured with softwood mixture produced 12.0 U/ ㎖ for endo-β-1,4-gulcanase (EG), 116.68 U/ ㎖ for β-glucosidase (BGL), 18.82 U/ ㎖ for cello- biohydrolase (CBH), and 13.33 U/ ㎖ for β-xylosidase (BXL). These levels of BGL, CBH, and BXL activities were two to four folds more than enzyme activities of F. palustris cultured with rice straw. The optimum reaction conditions of cellulase-RS which produced by F. palustris with rice straw and cellulase-SW which produced by F. palustris with softwood mixture were pH 5.0 at 45°C and pH 5.0 at 50°C, respectively. The sugar conversion yield of cellulase-SW had the highest value of 40.6 ± 0.6% within 72 h when rice straw was used as substrate. By adding 0.1% Tween 20 (w/w-substrate), the sugar conversion yield of rice straw was increased to 44%, which was about four fifths sugar conversion yield of commercial enzyme, Celluclast 1.5L (Novozyme A/S). A low crystallinity and an intensive fibril surface observed by the scanning electron microscope may explain the high sugar conversion yield of rice straw. Keywords : enzymatic hydrolysis, Formitopsis palustris, cellulase, rice straw, carbon source