Kinds Of Cellulases Research Articles

Enzymatic formulation strategies unlock highly-efficient saccharification of distinct pretreated corncobs

Optimization of the composition of enzyme cocktail is an effective way to improve the hydrolytic efficiency and reduce the cost during enzymatic hydrolysis of lignocellulose. Herein, the optimal enzymes ratios of enzyme cocktails for efficient hydrolysis of corncobs with distinct pretreatments were quantitatively formulated. As the importance of pretreatment type on xylan extraction, the optimized cellulase/hemicellulase ratios for NaClO2, H2SO4 and steam explosion treated corncob were 1:1.5, 1:0.15 and 1:0.75, respectively. Meanwhile, different requirements for types of cellulases are also determined: endoglucanase and cellobiohydrolase were key enzymes in the hydrolysis of NaClO2 and H2SO4 treated corncobs, respectively, while the synergy of three kinds of cellulases was preferred for steam-exploded corncob. Furthermore, based on the optimized cellulase/hemicellulase ratios, cellulases proportions for hydrolyzing the corncobs pretreated with distinctive methods were determined. Notably, by the enzymatic formulation strategy, 100% yields of glucose and xylose were obtained simultaneously from NaClO2 treated corncob, which was up to 70.1% and 80.1% at high solid loading (20%), respectively. Without detoxification of hydrolysate, high isopropanol yield of 0.45 g/g (of dry matter) was achieved. Hence, this study demonstrated a low cost and high efficient conversion strategy for prospective high-value utilization of lignocellulose.

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.

Visualization of Functional Structure and Kinetic Dynamics of Cellulases.

Cellulose is the most abundant carbohydrate on earth and hydrolyzed by cellulases in nature. During catalysis, cellulase transfers protons to and from the oxygen atoms of the glycosidic bond and a water molecule. Since cellulose is an insoluble polymer, some kinds of cellulases, with high activity toward crystalline cellulose, move on the crystal surface with continuous hydrolysis of the molecular chain. In addition, binding and dissociation on/from the crystal surface are also important elementary steps of the reaction cycle. Recently, these interesting features of cellulases can be directly analyzed, due to the development of visualization techniques. In this chapter, we introduce (1) visualization of the protonation state of the catalytic residue by neutron crystallography, (2) visualization of processive movement on the crystal surface by high-speed atomic force microscopy , and (3) visualization of binding and dissociation events by single-molecule fluorescence microscopy.

Expression and Characteristics of an Endoglucanase from Trichoderma atroviride (TaEGII) in Saccharomyces cerevisiae.

Endoglucanase secreted by the fungus Trichoderma atroviride is a kind of cellulase. An endoglucanase gene egII was cloned from T. atroviride AS3.3013 and expressed in Saccharomyces cerevisiae INVScI. The open reading frame of the egII gene was composed of 1257bp, encoding 418 amino acids with a molecular weight of 44.23kDa plus a signal peptide of 21 amino acids. Based on sequence similarity, TaEGII belonged to the glycosyl hydrolase family 5. Expression of the egII gene in T. atroviride AS3.3013 can be induced by microcrystalline cellulose (MCC), bran, carboxymethyl cellulose (CMC), rice straw, and corn stalk but is inhibited by glucose. A highly efficient integrated expression vector (pYPIGH-B includes a sequence of the α-mating factor signal peptide (MF-α)) was constructed. The enzymatic activity of the supernatant of recombinant yeast YPIGH-B3 was 1.29 times higher than that of YES2-egII, demonstrating that the MF-α can significantly improve the expression of the recombinant EGII in S. cerevisiae. The recombinant endoglucanase TaEGII produced by S. cerevisiae showed maximum activity at pH 5.0 and temperature 60°C. Under these conditions, the Km and Kcat values for Avicel and raffinose hydrolysis were 1.22×10-2mgml-1, 9.09×10-2s-1 and 1.06×10-2mgml-1 , 9.18×10-2s-1, respectively. The enzymatic activity of recombinant TaEGII was stable when incubated from 40 to 60°C for 1h. It was stable in a wide range of pH (4.0-7.0) and sensitive to various metal ions. Transgenic yeast strain YPIGH-B3 might be applied to cellulosic ethanol production.

Gene Cloning and Expression of Cellulase of Bacillus amyloliquefaciens Isolated from the Cecum of Goose

ABSTRACTA kind of bacteria secreting cellulase and showing probiotic attributes was isolated from the cecum of goose and identified as Bacillus amyloliquefaciens by analysis of 16S rRNA gene sequence and named as B. amyloliquefaciens S1. In vitro assays, the enzymatic activity of the strain was determined by the reducing-sugar method, and the proper culture conditions of producing cellulase and some properties of the cellulase were investigated. The cultural mixture of the bacteria had a high cellulase activity of 1.25 U/mL. In order to improve the utilization rate of the cellulase, some properties of the cellulase were studied. The best reaction pH of the enzymes was 7.0 and the optimum reaction temperature was 60°C. The enzyme was a kind of neutral cellulase that possessing strong resistance against heat and acidity. It showed high activity to absorbent cotton, soybean meal, and filter paper. Meanwhile, a gene encoding a kind of cellulase was cloned and prokaryotic expressed in Escherichia coli. The gene had 1500 bp in length, encoding a protein of 55 kDa, which was confirmed by SDS-PAGE and Western blotting. This study explored the possibility of degrading ability of bacteria with its probiotic attributes to enhance digestibility of the feed and gut health of animal. It also provided some basis for its further functional analysis and practical application as a microbial preparation for the breeding.

Development of cellulosic ethanol production process via co-culturing of artificial cellulosomal Bacillus and kefir yeast

A novel dual-microbe Bacillus/yeast co-culture system is developed for cellulosic bioethanol production. A recombinant cellulosomal Bacillus subtilis that carries eight cellulosomal genes of Clostridium thermocellum, including one scaffolding protein gene (cipA), one cell-surface anchor gene (sdbA), two exo-glucosidase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ) was constructed. The partner microbes for the dual-microbe combination are the wild type kefir yeast Kluyveromyces marxianus KY3, K. marxianus KY3-NpaBGS, which carries a β-glucosidase (NpaBGS) gene from rumen fungus, and the K. marxianus KR5 strain, that harbors endoglucanase (egIII), exo-glucanase (cbhI) and NpaBGS genes. All three Bacillus/yeast co-culture systems could achieve the cellulose saccharification and ethanol conversion simultaneously better than KR5 alone. The combination of Bacillus/KY3-NpaBGS outperformed that of Bacillus/KY3, as they could produce β-glucosidase enzyme for the system. Although, KR5 produces two more kinds of cellulases than KY3-NpaBGS. Bacillus/KR5 could not perform better than Bacillus/KY3-NpaBGS. Our results suggest that the dual-microbe Bacillus/yeast co-culturing system could leverage the advantages from both microbes and have a great potential for integrating into consolidated bioprocessing system.

Biological Conversion of Corncob Residues of Xylose Manufacture to L-Lactic Acid

The aim of this research is to study the saccharification of corncob residues of xylose manufacture by enzymes and turn it to L-lactic acid by fermentation. Corncob residues of xylose manufacture is one kind of lignocelluloses composed of 48.5% cellulose, 21.3% lignin, and 23.5% hemicellulose. As one of the most widely used organic acids in industry and the precursor of PLA, a degradable plastic, L-lactic acid is a very important material. In this study, six kinds of cellulases and one kind of β-glucosidase produced by different companies were studied to obtain high yield sugars needed for L-lactic acid fermentation. Results showed that composite of different enzymes could improve the catalysis effects. Mixture of cellulose F3: cellulose F4: β-glucosidase at the ratio of 2:4:9 engendered a high synergistic effect in hydrolysis. Also, the main factors influencing the hydrolysis of corncob residues were investigated. The appropriate reaction conditions are sodium acetate buffer 0.05-0.1mol/L, pH 5.0-5.5, concentration of corncob residues 15%, enzyme concentration 97U for 1g substrate, reaction temperature 50°C and the shaker speed 140 r/min. After 96h reaction, the concentration of glucose could reach as high as 5.5%. In fermentation, 4.48% of L-lactic acid was produced in 24 hours utilizing hydrolysis sugars as the carbon source, and the percent conversion of glucose to L- lactic acid was 81.5%.

Hydrolytic Enzyme of Cellulose for Complex Formulation Applied Research

To improve the enzymatic hydrolytic efficiency and reduce the supplementation of enzymes, the mixture designed experimental approach was used to optimize the composition of enzyme mixture and promote the hydrolysis of ball-milled corn stover. From the experimental results, a synergistic effect was found when combinations of the three enzymes, two kinds of cellulases and a kind of xylanase, were used. The optimal hydrolysis of pretreated corn stover accorded with enzymes activity ration of FPU/CMCase/β-glucosidase/xylanase = 4.4:1:75:829, and the hydrolysis efficiency of corn stover increased significantly compared with using individual enzyme. The results indicated that the mixture design experiment could be an effective tool for optimized enzyme mixture for lignocellulose hydrolysis.