Cellulase Protein Research Articles

The Phylogenomic Characterization of Planotetraspora Species and Their Cellulases for Biotechnological Applications.

This study aims to evaluate the in silico genomic characteristics of five species of the genus Planotetraspora: P. kaengkrachanensis, P. mira, P. phitsanulokensis, P. silvatica, and P. thailandica, with a view to their application in therapeutic research. The 16S rRNA comparison indicated that these species were phylogenetically distinct. Pairwise comparisons of digital DNA-DNA hybridization (dDDH) and OrthoANI values between these studied type strains indicated that dDDH values were below 62.5%, while OrthoANI values were lower than 95.3%, suggesting that the five species represent distinct genomospecies. These results were consistent with the phylogenomic study based on core genes and the pangenome analysis of these five species within the genus Planotetraspora. However, the genome annotation showed some differences between these species, such as variations in the number of subsystem category distributions across whole genomes (ranging between 1979 and 2024). Additionally, the number of CAZYme (Carbohydrate-Active enZYme) genes ranged between 298 and 325, highlighting the potential of these bacteria for therapeutic research applications. The in silico physico-chemical characteristics of cellulases from Planotetraspora species were analyzed. Their 3D structure was modeled, refined, and validated. A molecular docking analysis of this cellulase protein structural model was conducted with cellobiose, cellotetraose, laminaribiose, carboxymethyl cellulose, glucose, and xylose ligand. Our study revealed significant interaction between the Planotetraspora cellulase and cellotetraose substrate, evidenced by stable binding energies. This suggests that this bacterial enzyme holds great potential for utilizing cellotetraose as a substrate in various applications. This study enriches our understanding of the potential applications of Planotetraspora species in therapeutic research.

Fungal cellulolytic enzyme complex immobilized on chitosan-functionalised magnetic nanoparticles for paddy straw saccharification

Utilization of biopolymeric functionalized nanocomposites for enzyme immobilization has been actively explored for their potential role as catalysts in sustainable agro-residue valorization models. In the present study, cellulase immobilization on control and chitosan functionalized magnetic nanoparticles (c-MNP & Ch-MNP, respectively) was investigated. A cellulase binding efficiency of 67.50% on c-MNP and 74.06% on Ch-MNP was achieved. The enzyme immobilization on nano-supports was confirmed using electron microscopic and spectroscopic techniques. The immobilized cellulase (E-Ch-MNP) exhibited carboxymethyl cellulase, cellobiase and avicelase specific activities of 48.68, 25.98 and 33.80 nmol min−1 mg−1 protein. The optimum pH for free and immobilized enzyme were reported to be pH 5 and 6, respectively. An increased thermal stability for E-Ch-MNP was observed over a broader temperature range with a peak relative enzyme activity at 60℃. The binding constant (Km= 5.00 mg ml−1) and maximal rate of reaction (Vmax= 0.12 µmol min−1 ml−1) were also determined for E-Ch-MNP. Molecular docking studies determined the flexibilities of both the receptor cellulase protein and the ligand immobilization support molecules with respect to the binding energy and inhibition constants, reflecting their desired conformation and stability. Maximum reducing sugar content of 242.14 mg g−1 of pre-treated straw was reported at 96 h of incubation for E-Ch-MNP with a saccharification efficiency of 35.78%. The retained relative activity of immobilized cellulase was 82.83, 75.17, 44.61 and 27.95% of initial activity after second, third, fourth and fifth cycle, respectively. Therefore, immobilization of cellulase on chitosan coated magnetic nanoparticles holds promise for further optimization for improved cellulose hydrolysis.

Integrative transcriptome and proteome analyses of Trichoderma longibrachiatum LC and its cellulase hyper-producing mutants generated by heavy ion mutagenesis reveal the key genes involved in cellulolytic enzymes regulation

BackgroundThe major challenge of facing the efficient utilization of biomass is the high cost of cellulolytic enzyme, while the Trichoderma longibrachiatum plays an essential role in the production of industrial enzymes and biomass recycling.ResultsThe cellulase hyper‑producing mutants of LC-M4 and LC-M16 derived from the wild type T. longibrachiatum LC strain through heavy ion mutagenesis exhibited the high-efficiency secretion ability of cellulase and hemicellulose. The FPase activities of LC-M4 (4.51 IU/mL) and LC-M16 (4.16 IU/mL) mutants increased by 46.91% and 35.5% when compared to the LC strain, respectively. Moreover, these two cellulase hyper-producing mutants showed faster growth rate on the cellulosic substrates (Avicel and CMC-Na) plate than that of LC strain. Therefore, an integrative transcriptome and proteome profiling analysis of T. longibrachiatum LC and its cellulase hyper‑producing mutant LC-M4 and LC-M16 were employed to reveal the key genes involved in cellulolytic enzymes regulation. It was showed that the transcriptome and proteome profiles changed dramatically between the wild strain and mutant strains. Notably, the overlapped genes obtained from integrative analysis identified that the protein processing in ER involved in protein secretory pathway, starch and sucrose metabolism pathway and N-glycan biosynthesis pathway were significantly changed both in cellulase hyper-producing mutants and thereby improving the enzyme secretion efficiency, which maybe the main reason of cellulase hyper-production in LC-M4 and LC-M16 mutants. In addition, the three DEGs/DEPs (PDI, Sec61, VIP36) related with protein secretion in ER and two DEGs/DEPs (OST, MOGS) related with N-glycan biosynthesis were identified as key candidate genes participating in enzyme protein biosynthesis and secretion.ConclusionsIn this study, a hypothetical secretory model of cellulase protein in filamentous fungi was established on the basis of DEGs/DEPs and key genes identified from the omics analysis, which were of great guidance on the rational genetic engineering and/or breeding of filamentous fungi for improving cellulase production.

Isolation of a Heat-Resistant Aspergillus Fumigatus and Identification of its Cellulase

Cellulose-degrading fungi have the characteristics of large enzyme production, high enzyme activity, comprehensive enzyme composition, and most of them are secreted and extracted extracellularly. Therefore, studies on cellulase-producing bacteria at home and abroad have focused on fungi. Due to its thermal stability, the high temperature resistant cellulase can meet the high temperature conversion conditions in production, which is beneficial to the improvement of cellulose degradation efficiency. This chapter mainly carried out the separation screening and identification of heat-resistant cellulase-producing fungi, and the analysis of cellulase protein components and concentrations in fermentation products by Nano LC-ESI-MS/MS.

Statistical optimization of lignocellulosic waste containing culture medium for enhanced production of cellulase by Bacillus tequilensis G9

The ever increasing energy demands of modern civilization and rapidly dwindling fossil fuels point towards a renewable substitute like biofuels. However, higher costs associated with biofuel productions is the major bottleneck for its commercialization. The present study demonstrates the use of a statistical approach called response surface methodology (RSM) to investigate the optimum parameters for maximum production of cellulase by Bacillus tequilensis G9. The Plackett–Burman design (PB) of the RSM analysis indicated grass straw (GS) concentration, pH, FeSO4, inoculum, MgSO4, incubation period and NH4Cl as significant variables that influence the cellulase production. Further, to propose the best medium for the maximum production of cellulase by B. tequilensis G9, the most influential parameters, namely concentrations of GS as substrate, FeSO4, pH, inoculum size, etc. were fine-tuned by central composite design (CCD) involving four factors and five levels. The CCD analysis demonstrated 8% substrate concentration, 1.5% of inoculum along with 10 ppm FeSO4 and a pH of 5.5 in media as optimum conditions for highest enzyme production. The field emission scanning electron microscopic analysis of the treated GS showed structural alterations depicting significant deconstruction caused by B. tequilensis G9. The yield of the partially purified cellulase proteins were found to be 21% revealing molecular mass between 30 and 97 kDa. The enhanced cellulase production by B. tequilensis G9 demonstrated in our study brands its applications in many industrial processes like biorefinery, biofuels, etc.

Production and optimization of high grade cellulase from waste date seeds by Cellulomonas uda NCIM 2353 for biohydrogen production

Production of high grade cellulolytic enzymes from waste agricultural biomass would valorise these wastes to valuable products as well as avoid the pollution problems associated with landfilling of the biomass. In the present study, waste date palm (Phoenix dactylifera) seeds were valorised for cellulase production from Cellulomonas uda NCIM 2353 and for its subsequent usage in biohydrogen production. Optimization of key operational parameters such as date seed concentration, xylose, casein and initial media pH were performed using central composite design to obtain the maximum enzyme yield. The optimum values obtained were (g/L): date seed concentration 30.65, xylose concentration 0.55, casein 7.00 and pH 7.40 for a determination coefficient of 0.999. The results demonstrated a higher prediction accuracy of response surface methodology as the cellulase activity increased six fold (175.96 IU/mL) after optimization. The optimum pH and temperature of purified cellulase was 7 and 50 °C respectively where the enzyme retained nearly 80% of activity upto 180 min. Enzymatic hydrolysis studies showed that a high saccharification efficiency of 60.5% was obtained for acid pretreated sugarcane bagasse by the indigenous cellulase, equivalent to the performance of commercial cellulase. Further, the as-obtained reducing sugars were decomposed by Clostridium thermocellum to produce biohydrogen of maximum concentration 187.44 mmol/L at end of 24 h of fermentation. Results show that date seed substrate based cellulase protein can be employed for industrial processes of biohydrogen production.

Promoting enzymatic hydrolysis of lignocellulosic biomass by inexpensive soy protein

BackgroundLiquid hot water (LHW) pretreatment has been considered as one of the most industrially viable and environment-friendly methods for facilitating the transformation of lignocelluloses into biofuels through biological conversion. However, lignin fragments in pretreatment hydrolysates are preferential to condense with each other and then deposit back onto cellulose surface under severe conditions. Particularly, lignin tends to relocate or redistribute under high-temperature LHW pretreatment conditions. The lignin residues on the cellulose surface would result in significant nonproductive binding of cellulolytic enzymes, and therefore negatively affect the enzymatic conversion (EC) of glucan in pretreated substrates. Although additives such as bovine serum albumin (BSA) and Tween series have been used to reduce nonproductive binding of enzymes through blocking the lignin, the high cost or non-biocompatibility of these additives limits their potential in industrial applications.ResultsHere, we firstly report that a soluble soy protein (SP) extracted from inexpensive defatted soy powder (DSP) showed excellent performance in promoting the EC of glucan in LHW-pretreated lignocellulosic substrates. The addition of the SP (80 mg/g glucan) could readily reduce the cellulase (Celluclast 1.5 L®) loading by 8 times from 96.7 to 12.1 mg protein/g glucan and achieve a glucan EC of 80% at a hydrolysis time of 72 h. With the same cellulase (Celluclast 1.5 L®) loading (24.2 mg protein/g glucan), the ECs of glucan in LHW-pretreated bamboo, eucalyptus, and Masson pine substrates increased from 57%, 54% and 45% (without SP) to 87%, 94% and 86% (with 80 mg SP/g glucan), respectively. Similar effects were also observed when Cellic CTec2, a newer-generation cellulase preparation, was used. Mechanistic studies indicated that the adsorption of soluble SP onto the surface of lignin residues could reduce the nonproductive binding of cellulolytic enzymes to lignin. The cost of the SP required for effective promotion would be equivalent to the cost of 2.9 mg cellulase (Celluclast 1.5 L®) protein (or 1.2 FPU/g glucan), if a proposed semi-simultaneous saccharification and fermentation (semi-SSF) model was used.ConclusionsNear-complete saccharification of glucan in LHW-pretreated lignocellulosic substrates could be achieved with the addition of the inexpensive and biocompatible SP additive extracted from DSP. This simple but remarkably effective technique could readily contribute to improving the economics of the cellulosic biorefinery industry.

Effect of alkaline lignin modification on cellulase\u2013lignin interactions and enzymatic saccharification yield

BackgroundThe lignin can compete for binding cellulase enzymes with cellulose fibers and decrease the accessibility of enzymes to carbohydrates. The competitive adsorption of cellulase to lignin mainly depended on the chemical structure of lignin. The post-pretreatment can decrease the lignin content and modify the lignin structure of pretreated substrates, which reduced the lignin inhibition on enzymatic saccharification. Therefore, the post-treatment by modifying the lignin structure would attract considerable attention for weakening the cellulase–lignin interactions.ResultsThree modified lignins, including sulfonated lignin (SL), oxidized lignin (OL), and carboxylated lignin (CL), were prepared from alkali lignin (AL) and their structures and physicochemical properties were characterized using FTIR, NMR, XPS analysis, zeta potential, and contact angle, respectively. Compared to AL, three modified lignin preparations exhibited the decrease in contact angle by 61–70% and phenolic hydroxyls content by 17–80%, and an obvious increase of negative charges by about 21–45%. This was mainly due to the drop of condensation degree and the incorporation of carboxylic and sulfonic acid groups into modified lignins. Langmuir adsorption isotherms showed that the affinity strength between cellulase and modified lignins significantly reduced by 54–80%. Therefore, the 72 h hydrolysis yield of Avicel with SL, OL, and CL was 48.5, 51.3, and 49.4%, respectively, which was increased 8–15.3% than that of Avicel with AL, 44.5%. In the enzymatic hydrolysis of bamboo biomass, the glucose yield at 5 d was 38.5% for AS-P. amarus, 15.4% for AO-P. amarus and 21.4% for AC-P. amarus, respectively, which were 1.4–3.5 times of alkali pretreated P. amarus.ConclusionsThe post-treatment can weaken the nonproductive adsorption between lignin and cellulase proteins and improve the enzymatic saccharification efficiency. This study will provide a conceptual combination of pretreatment technologies and post-pretreatment by modifying lignin structure for reducing the cellulase–lignin interaction.

Modeling of Oxygen Delignified Wheat Straw Enzymatic Hydrolysis as a Function of Hydrolysis Time, Enzyme Concentration, and Lignin Content

Enzymatic hydrolysis is one of the most expensive operations of producing lignocellulosic ethanol, primarily due to high enzyme costs. Enzyme loadings must be reduced, and a well-developed kinetic model that can be easily implemented in process simulation software would greatly assist in determining optimum processing conditions. Oxygen-delignified wheat straw with different lignin contents was subjected to enzymatic hydrolysis at two different enzyme loadings for 72 h. Glucan conversion increased with increasing enzyme loading, decreasing lignin content, and decreasing solids concentration. By measuring total protein concentration and predicting the Novozyme 188 protein concentration, it was possible to calculate the cellulase protein concentration as a function of time. This work is the first report of a mass-based kinetic model capable of predicting glucose production during enzymatic hydrolysis of oxygen-delignified wheat straw, at different cellulases loadings (20 and 40 filter paper units/g glucan), lignin contents (5 and 9 wt%), and solids concentrations (5 to 10 wt% dry basis). The presented hydrolysis model includes a novel lignin factor to describe the amount of cellulases irreversibly adsorbed on lignin. The lignin factor also links glucose production during enzymatic hydrolysis to pretreatment severity. Mass transfer limitations present at 10 wt% solids were accounted for using a diffusion factor. Due to the model's simple solution and use of only five parameters, it can be easily implemented in process simulations.

Utilizing cellulase as dispersant to prepare stable nano-pigment suspension and investigating its coloring performance on cotton fabrics

In this research, a novel nano-pigment suspension was successfully prepared in an ultrasonic disruptor using crude cellulase as dispersant. The colloidal and rheological properties of this cellulase-based suspension such as zeta potentials, particle distribution and apparent viscosity were analyzed. The measurement of zeta potentials showed that the surface charges of pigment were adjustable along with the acid–base properties of external environment due to the amphoteric character of cellulase proteins. Subsequently, its stability to high-speed centrifuge, freeze–thaw treatment and ion intensity was carefully investigated. Thermo-gravimetric analysis (TGA) was conducted to demonstrate the coverage of pigment particles by cellulase proteins. Furthermore, X-ray diffraction (XRD) characterization was utilized to disclose the combined impacts of cellulase and ultrasonic power on the crystal structures of pigment. At last, the coloring performance of this charge-adjustable nano-pigment suspension on cotton substrates was evaluated by measuring their K/S values and color fastness. Scanning Electron Microscope (SEM) was carried out to give a direct observation of the nano-pigment distributed on cotton surfaces.

The influences of enzymatic processing on physico-chemical and pigment dyeing characteristics of cotton fabrics

First, a crude cellulase was used to treat cotton fabrics to investigate its influences on the physicochemical properties of cotton. The FTIR and XRD analyses both confirmed the enzymatic treatment could increase the crystallinity of cotton, especially at a higher cellulase dosage. Once treated, the number of dissociable groups (–COOH) in cotton decreased, while that of the reducing groups (–CHO) increased. Second, copper phthalocyanine (CuPc) was selected to prepare an anionic nanoscale pigment dispersion to detect its dyeability on different cotton samples. It was concluded that the enzymatic hydrolysis itself had no significant impacts on the pigment dyeing performance. However, cellulase protein still stayed on the cotton surface after treatment and produced an enhancement effect on the pigment uptake due to strong hydrophobic interactions between them. This could be verified by K/S measurement and SEM observations.

Evaluation of hollocelulase production by Lentinula edodes (Berk.) Pegler during the submerged fermentation growth using RSM.

The cellulase proteins have a great importance in the enzymatic hydrolysis of woody biomass. Despite of costs being a major concern, it has been a stimulus to study basidiomycetes biochemical properties which degrade lignocellulosic material and have prompted the processes' study for obtaining cellulolytic enzymes in fungi. The objective of this research was to evaluate the effects of the initial nitrogen content on (ammonium sulfate) and on sugar cane bagasse, which hereby, acts as an inducer of hydrolytic enzymes to produce cellulases and xylanases, using three Lentinula edodes (Berk.) Pegler strains as a transformation agent. A factorial design with 22 replications in the central point was conducted, varying concentrations of ammonium sulfate and sugar cane bagasse. The submerged cultures carried out in synthetic culture medium and incubated at 25°C for 7 days on an orbital shaker at 150 rpm. The total protein and cellulase activity as endoglucanase, exoglucanase and β-glucosidase and the xylanase was also determined. The results showed that the production of hydrolytic enzymes was stimulated by the presence of high concentrations of sugar cane bagasse (30g/L), characterizing it as an inducer due to the demonstrated proportional relationship. Thus, ammonium sulfate acted as a reducing agent in the synthesis of enzymes, being the low concentrations (0.1g/L) indicated for the enzyme production system under study. Among the studied strains, the EF52 showed higher activity for xylanase, endoglucanases, β-glucosidase and also protein.

RNAi Knockdown of Potent Sugar Sensor in Cellulase-Producing Fungus Acremonium cellulolyticus

A potent sugar sensor gene (g9105) was screened from the genomic data of the cellulase-producing fungus Acremonium cellulolyticus. The transcriptional level of g9105 in the SA49 transformant, which carried the knockdown RNA interference (RNAi) construct, was less than 10% compared with the parental YP-4 strain. The hairpin-type RNAi construct could be useful for this fungal gene knockdown. Changes in cellulase productivity and protein secretion between these two strains were not observed. The numbers of hyphal tips at subapical branching site were counted for the SA49 and YP-4 strains incubated with potato-dextrose medium at 30 °C for 4 days. The hyphal branching ratio of the SA49 strain was higher than that of the YP-4 strain. The present results suggest that the potent sugar sensor gene in A. cellulolyticus could be related with hyphal branch formation.

Selection of optimumTrichodermaspecies for cellulase activity and glucose production from wheat crust

Cellulase enzyme and glucose are important industrial bioproducts which can be obtained from cheap agrowastes. The aim of the present study was to select optimum Trichoderma species for cellulase activity and production of glucose from wheat crust. Three different strains of this fungus i. e. T. koningi 5139, T. longibrachiatum 5140 and T. reesei 5142 were selected. The conditions were optimized by using wheat crust and culturing Trichoderma species with solid state fermentation method. Different incubation times (3–8 days), temperatures (26, 28, 30, 32 and 34°C) and pHs (3.0–9.0) were experimented for the production of cellulase and glucose. Cellulase activity, glucose and protein were measured using standard filter paper activity, enzymatic and Lowry methods in raw enzymatic solution of the cultures, respectively. Cellulase activity and produced glucose were higher in T. reesei compared with two other strains in the same condition (P<0.05). Our results demonstrated that T. reesei was the optimum strain. Also, best culture conditions were 96 h of incubation at 30°C and pH 6.0 for giving highest cellulase activity and produced glucose.

Utilizing cellulase as a hydrogen peroxide stabilizer to combine the biopolishing and bleaching procedures of cotton cellulose in one bath

In this research, the stabilization effect of cellulase on the decomposition of hydrogen peroxide was investigated for the first time. It was concluded that, regardless of the decomposition mechanism, the cellulase protein could contribute significantly to peroxide stability. This effect stems from the formation of molecular hydrogen bonding between peroxide and cellulase protein or direct sequestering of free metal ions by amino acids in cellulase. Furthermore, based on this stability, a combined biopolishing and peroxide bleaching protocol was developed to improve cotton quality more efficiently. Afterwards, physicochemical properties such as the weight and strength loss, water absorbency, and carbonyl and carboxyl group content of treated cotton cellulose were measured to show the feasibility of the new method. Fourier-transform infrared (FT-IR) and X-ray diffraction (XRD) analyses indicated that the crystallinity index of cotton was increased due to the preferential hydrolysis of amorphous cellulose by cellulase.

Modified alumina nanofiber membranes for protein separation

Large-scale purification/separation of bio-substances is a key technology required for rapid production of biological substances in bioengineering. Membrane filtration is a new separation process and has potential to be used for concentration (removal of solvent), desalting (removal of low molecular weight compounds), clarification (removal of particles), and fractionation (protein-protein separation). In this study, we developed an efficient membrane for protein separation based on ceramic nanofibers. Alumina nanofibers were prepared on a porous support and formed large flow passages. The radical changes in membrane structure provided new ceramic membranes with a large porosity (more than 70%) due to the replacement of bulk particles with fine fibers as building components. The pore size had an average of 11nm and pure water flux was approximately 360Lh−1m−2bar−1. Further surface modification with a self-assembled monolayer of (3-aminopropyl) triethoxysilane enhanced the membrane filtration properties. Characterization with SEM, FTIR, contact angle, and proteins separation tests indicated that the fibril layers uniformly spread on the surface of the porous support. Moreover, the membrane surface was changed from hydrophilic to hydrophobic after silane groups were grafted. It demonstrated that the silane-grafted alumina fiber membrane can reject 100% BSA protein and 92% cellulase protein. It was also able to retain 75% trypsin protein while maintaining a permeation flux of 48Lh−1m−2bar−1.

Adsorption of proteins involved in hydrolysis of lignocellulose on lignins and hemicelluloses

Protein adsorption onto eight lignocellulosic substances (six lignin preparations and two hemicelluloses) was investigated at pH 4.8 and at two different temperatures (4°C and 45°C). The kinetics of the adsorption of cellulase, xylanase, and β-glucosidase were determined by enzyme activity measurements. The maximum adsorption capacities, the affinity constants and the binding strengths varied widely and were typically higher for the lignins than for the carbohydrates. As indicated by BET and gel permeation chromatography, different substances had widely different surface area, pore size, weight average molecular weight, and polydispersity index, but these properties were difficult to relate to protein binding. In most cases, an increase in temperature from 4°C to 45°C and a low content of carboxylic acid groups, as indicated by Fourier-Transform Infra-Red (FTIR) spectroscopy, resulted in increased protein adsorption capacity, which suggests that hydrophobic interactions play an important role.

Plant growth promoting potential and soil enzyme production of the most abundantStreptomycesspp. from wheat rhizosphere

To evaluate the plant growth promotion (PGP) potential and soil enzyme production under solid state fermentation (SSF) by most abundant Streptomyces spp. isolated from the wheat rhizosphere and to evaluate their effect on plant growth parameters. Actinomycetes were isolated from wheat rhizosphere and screened for PGP activities. Three actinomycete isolates having significantly higher PGP activities (Streptomyces rochei IDWR19, Streptomyces carpinensis IDWR53, Streptomyces thermolilacinus IDWR81) were selected. The soil enzymes production potential of these isolates using soil extract and wheat straw under ssf was assessed. Utilization of soil extract as a fermentation medium for soil enzyme production by Actinomycetes has been reported first time in this study. Maximum chitinase (S. rochei IDWR19 12·2 U mg(-1) protein) and phytase activity (S. carpinensis IDWR53 5·2 U mg(-1) protein) was produced on 7th day of incubation, whereas maximum alkaline protease (S. rochei IDWR19 3·2 U mg(-1) protein) was produced on 6th day of incubation. For cellulase (S. rochei IDWR19 7·4 U mg(-1) protein) and invertase (S. carpinensis IDWR53 451 U mg(-1) protein) maximum activity was observed on 4th as well as 5th day of incubation. On the basis of PGP activity and enzyme production, two actinomycete isolates (S. rochei IDWR19 and S. thermolilacinus IDRWR81) were selected for plant growth experiment. An increase of 12·2 and 24·5% in shoot length of plants inoculated with S. rochei IDWR19 and S. thermolilacinus IDWR81 was observed, respectively. A similar increase in biomass of 1·8- and 2·3-fold was also recorded for the two isolates, respectively. It could be concluded that Streptomyces sp. with high PGP activities and soil enzyme production capability significantly improved growth and development of wheat cv. The abundant Actinomycetes obtained in this study (S. rochei IDWR19 and S. thermolilacinus IDWR81) are rhizosphere competent and effective strains.

Toxic misfolding of Arabidopsis cellulases in the secretory pathway of Pichia pastoris

Plants produce a large number of cellulases that are either secreted or anchored in the plasma membrane where they likely function in various aspects of cellulose synthesis, modification and degradation during plant growth and development. Very few of these enzymes have been characterized in any detail, however. Here we attempted to produce two Arabidopsis modular cellulases, which contain a catalytic domain belonging to glycoside hydrolase family 9 (GH9) and a carbohydrate binding module (CBM), in the yeast Pichia pastoris. Neither of the intact modular enzymes was detectably produced, although the independently expressed GH9 catalytic domain of one enzyme was secreted when the protein was expressed at low temperature. Expression of intact and truncated cellulases at the standard temperature caused extensive cell lysis, with release of high concentrations of endogenous proteins into the culture medium. Cell lysis appeared to result from misfolding of cellulase proteins within the Pichia secretory pathway. The toxicity of these misfolded cellulases potentially could be exploited to derive host strains with enhanced capability to fold recombinant secretory proteins.

Effects of low intensity ultrasound on cellulase pretreatment

This research was to explore the mechanism of ultrasonic impact on free cellulase activity and immobilize cellulase activities. The highest free cellulase activity was achieved when the sample was treated with low intensity ultrasound at 15W, 24kHz for 10min, under which the enzyme activity was increased by 18.17% over the control. Fluorescence and CD spectra revealed that the ultrasonic treatment had increased the number of tryptophan on cellulase surface slightly, with the deformation of certain number of α-helix structure and increase of random coil content in cellulase protein. The highest immobilized cellulase activity was achieved when the sample was treated with low intensity ultrasound at 60W, 24kHz for 10min, under which the enzyme activity was increased by 24.67% over the control. Scanning electron microscopy revealed that the ultrasonic treatment had increased the surface area of immobilized cellulase.