Both phytase and endoglucanase are additives in feed for mono-gastric animal known for their effects. Recombinant vector pPICZalpha-EG was constructed and transformed to GS115-phyA, a Pichia pastoris strain that had integrated with phytase gene, generating GS115-phyA-EG. Both phytase and endoglucanase activities in the supernatant were determined after methanol induction of GS115-phyA-EG. Phytase and endoglucanase activity reached 39.4% and 56.2% activity compared to GS115-phyA and GS115-EG, respectively. Properties of the mixed enzyme suggest that the optimal temperature and pH value be 55 degrees C and 5.5 respectively. Both phytase and endoglucanase showed greater than 80% activity across temperature ranges 45 degrees C to 55 degrees C and pH ranges 4.5 to 5.5. Expressing more than one enzyme in one system could save time and money during induced expression, and the mixed enzyme might apply for treating forge before feeding with poultry.
6-Phytase ; biosynthesis ; genetics ; Cellulase ; biosynthesis ; genetics ; Genetic Vectors ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Biofuels and bio-based chemicals from lignocellulosic biomass are sustainable, making them alternatives to petroleum-derived fuels and chemicals to address the challenges of the shortage of crude oil supply and climate change resulted from the overconsumption of petroleum-based products, particularly in China. However, high cost in liberating sugars from lignocellulosic biomass is still the bottleneck of the commercialization of biofuels and bio-based chemicals. In this article, the major components of cellulases and their synergistic role in the hydrolysis of pre-treated biomass is reviewed, followed by how to evaluate the enzymatic hydrolysis. With the elucidation of the underlying mechanism of the conformations of the enzyme molecules and their effectiveness in attacking cellulose substrate, more efficient enzymes are expected to be developed. Using the high production strain Penicillium decumbens, the on-site production of cellulases for cellulose ethanol production is discussed.
Biofuels ; Biomass ; Biotechnology ; methods ; trends ; Biotransformation ; Cellulase ; biosynthesis ; Hydrolysis ; Industrial Microbiology ; methods ; trends ; Lignin ; chemistry ; metabolism

The gelatinization process of the starch is replaced by unpolluted steam-pretreatment on the base of the Radix Puerariae rich in fiber and isoflavones. The production of ethanol and isoflavones by simultaneous saccharification and solid state fermentation (SSF) of steam-pretreatment Radix Puerariae is presented. The optimal technological conditions were obtained: Radix Puerariae being steam-pretreated at a saturated vapor pressure of 0.8 MPa for 3.5 min, glucoamylase(65 u/g), cellulase(1.5 u/g), 0.1%(NH4)2SO4, 0.1%KH2PO4 and activated yeasts being added in, and fermentation at 35-37 degrees C for 60 h. Under these conditions, the yield of ethanol and isoflavones from 100 g Radix Pureriae (dry basis) were 27.47 g and 4.43 g, respectively, the starch utilization rate was 95%. In comparison with the traditional fermentation technology, the simultaneous saccharification and SSF of steam-pretreatment Radix Puerariae is clean and energy-saving. It provides new way of the production of ethanol from the non-food starch material, and worthwhile to be explored and implemented in industry.
Cellulase ; metabolism ; Ethanol ; metabolism ; Fermentation ; physiology ; Glucan 1,4-alpha-Glucosidase ; metabolism ; Isoflavones ; biosynthesis ; Pueraria ; metabolism ; Steam ; Yeasts ; metabolism

The solid wastes of Chinese materia dedica industrialization represented by Salvia miltiorrhiza residues have a strong small-molecule bio-recalcitrance in the process of high-value utilization of biotransformation. Highly tolerant strains were bred to break bio-recalcitrance of Salvia miltiorrhiza residues and produce high-value added cellulose, which has a significant significance for recycling and industrial utilization of solid waste. In this study, a strain of fungus, Penicillium expansum SZ13, was found with small-molecule antibacterial substance tanshinone contained in Salvia miltiorrhiza residues by a biological method. The optimal enzyme production process and peak period of SZ13 were determined. It was found that SZ13 could maintain peak enzyme production for 5 days by degrading residues under the conditions of temperature 35 ℃, rotation speed 180 r·min~(-1), 5% of residues addition, and 5% seed solution addition. Meanwhile, the ability of SZ13 to degrade the enzyme production of multiple types of residues was explored. The results showed a high enzyme activity and stable enzyme production of SZ13 in the process of degrading residues. SZ13 could efficiently utilize various types of Chinese medicine residues, such as Salvia miltiorrhiza residues, to realize the high-value utilization of cellulose in multiple types of residues.
Cellulase/biosynthesis* ; China ; Drug Industry ; Drugs, Chinese Herbal ; Fermentation ; Materia Medica ; Penicillium/metabolism* ; Salvia miltiorrhiza ; Solid Waste

The gene (eg1) encoding for novel endoglucanase 1 was cloned previously from Chinese straw mushroom Volvariella volvacea. EG1 has high thermal stability and optimal pH at neutral and shows great potential in textile and paper industry applications. To improve the expression level of EG1 in Pichia pastoris, the increasing copy number of clone, and its high cell density fermentation in 3.2L fermenter for its high-level expression were investigated in this work. By electro-transformation of pPICZalphaB-egl into GS115EG11 integrated with single copy of eg1 gene, A resistant transformant with 3.8 times higher level expression than GS115EG11 was screened from YPDSZ plate containing 2000 microg/mL of Zeocin. The effect of initial cell density, pH and methanol on its expression and biomass accumulation was evaluated in shaking culture. Optimal EG1 production was observed when initial cell density OD600 was 5.0. EG1 production and biomass accumulation did not seem to vary when cells were induced at different pH values. Both of EG1 and cell density were found to increase with higher methanol concentrations, reaching 62.48 IU/mL and 31.7 (OD600) respectively after 120 h induction with 2.0% (V/V) methanol compared to 30.24 IU/mL and 17.79 (OD60) with 0.25% methanol induction. EG1 expression was further increased by 6.4 times higher than shaking culture after 95.5 hours induction with methanol in fed-batch fermentation, so totally 34 times higher than that for GS115EG11 was achieved by screening of high Zeocin resistant clone and high cell density fermentation. The production of EG1 with 543.36IU/mL CMC activity and 8.80mg/mL protein expression was obtained in Pichia pastoris.
Cellulase ; biosynthesis ; genetics ; Cloning, Molecular ; Culture Media ; Electroporation ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Fungal Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Volvariella ; enzymology ; genetics

Penicillium decumbens T. is an important filamentous fungus for the production of cellulases to effectively degrade lignocellulose for second generation biofuel production. In order to enhance the capability of Penicillium decumbens to produce cellulases, we constructed a creB (a deubiquitinating enzyme encoding gene) deletion cassette, and generated a creB knockout strain with homologous double crossover recombination. This mutation resulted in a detectable decrease of carbon catabolite repression (CCR) effect. The filter paper activity, endoglucanase activity, xylanase activity and exoglucanase activity of the deltacreB strain increased by 1.8, 1.71, 2.06 and 2.04 fold, respectively, when comparing with the parent strain Ku-39. A 2.68 fold increase of extracellular protein concentration was also observed. These results suggest that the deletion of creB results in CCR derepression. These data also suggest that CREB influences cellulase production of Penicillium decumbens. In generation, this study provides information that can be helpful for constructing cellulase hyper-producing strain.
Cellulase ; biosynthesis ; Endopeptidases ; genetics ; metabolism ; Gene Knockout Techniques ; Lignin ; metabolism ; Mutant Proteins ; metabolism ; Penicillium ; enzymology ; genetics ; Recombination, Genetic ; Ubiquitinated Proteins ; genetics ; Ubiquitination

This study aims to observe different factors which affected the bionic enzymatic hydrolysis of icariin into baohuoside I and to optimize the reaction conditions in order to provide research foundation for building a novel bionic enzymolysis drug delivery system. To simulate the environment in vivo, 37 degrees C was set as the temperature and artificial intestinal juice and gastric juice were selected as the buffer solutions. Taking the conversion of baohuoside I as index, the effects of the kinds of enzyme, enzyme activity, substrate concentration, reaction time, pancreatin in artificial intestinal juice and surfactant on the conversion of baohuoside I were investigated. The results showed that cellulase, beta-glucosidase and snailase were all inactive in artificial gastric juice and no baohuoside I generated. Pancreatin in artificial intestinal juice couldn't significantly influence the activity of beta-glucosidase or snailase (P > 0.05), but noticeably decrease the activity of cellulase (P < 0.05). In artificial intestinal juice, the conversion of baohuoside I was highest by using beta-glucosidase, and the optimum reaction conditions were determined as follows: enzyme activity 10 U x mL(-1), substrate concentration 1 mg x mL(-1), 3 g x L(-1) rhamnolipid and reaction time 3 h. Under this condition, the conversion of baohuoside I was 99.8%.
Animals ; Cellulase ; chemistry ; Flavonoids ; biosynthesis ; metabolism ; Hydrolases ; chemistry ; isolation & purification ; Hydrolysis ; Pancreatin ; chemistry ; Snails ; enzymology ; Surface-Active Agents ; chemistry ; beta-Glucosidase ; chemistry

In the storage of Radix Ophiopogonis, browning often happens to cause potential risk with regard to safety. Previously few reports investigate the browning of Radix Ophiopogonis. In this research, the causes and mechanisms of the browning of Radix Ophiopogonis were preliminarily elucidated. Content determination by high-performance liquid chromatography (HPLC) and spectrophotometry, enzyme activity determination by colorimetry, and morphological observation by electron microscopy were performed in the present study. Uniform design and three-dimensional response surfaces were applied to investigate the relationship between browning and storage factors. The cortex cell wall of browned Radix Ophiopogonis was ruptured. Compared with the normal Radix Ophiopogonis, cellulase and polyphenol oxidase enzymes were activated, the levels of 5-hydroxymethylfurfural (5-HMF), total sugars, and reducing sugars were increased, while the levels of polysaccharides and methylophiopogonanone A were decreased in browned Radix Ophiopogonis. The relationship between the storage factors and degree of browning (Y) could be described by following correlation equation: Y = - 0.625 4 + 0.020 84 × X3 + 0.001 514 × X1 × X2 - 0.000 964 4 × X2 × X3. Accompanied with browning under storage conditions, the chemical composition of Radix Ophiopogonis was altered. Following the activation of cellulase, the rupture of the cortex cell wall and the outflow of cell substances flowed out, which caused the Radix Ophiopogonis tissue to become soft and sticky. The main causes of the browning were the production of 5-HMF, the activation of polyphenol oxidase, Maillard reactions and enzymatic browning. Browning could be effectively prevented when the air relative humidity (HR), temperature, and moisture content were under 25% RH, 12 °C and 18%, respectively.
Carbohydrates ; biosynthesis ; Catechol Oxidase ; Cell Wall ; enzymology ; Cellulase ; Chromatography, High Pressure Liquid ; Food Storage ; methods ; Furaldehyde ; analogs & derivatives ; chemical synthesis ; Humidity ; Maillard Reaction ; Ophiopogon ; chemistry ; enzymology ; Temperature

Cellulases are relatively costly enzymes that are sold in large volumes for use in different industrial applications, and a significant reduction in cost will be important for their commercial use in biorefineries. The production of cellulase is a major factor in the hydrolysis of cellulosic materials. Hence it is essential to make the process economically viable. A strain (L-06) with high cellulase activity was screened from rice straw compost and classified as Penicillium decumbens by the analysis of its morphology and 18S rRNA gene sequences. Different conditions of liquid fermentation medium including nitrogen source, carbon source, surfactant, temperature, initial pH, inoculation quantity for the production of cellulase had been studied. The maximal beta-1, 4-glucosidase(BGL) activity was 1662 u/mL which is 1.49 times of the previous and the maximal exo-beta-1, 4-glucanases(CBH) activity was 2770 u/mL which is 1.36 times of the previous, cultured in the optimal condition for three days. And the maximal endo-beta-1, 4-glucanases (EG) activity was 18064 u/mL which is 1.87 times of the previous and the maximal filter paper enzyme(FPase) activity was 4035 u/mL which is 1.47 times of the previous, cultured in the optimal condition for four days. In the optimization experiments, the EG and CBH in the culture condition (pH10) maintained 70% and 43% activity. In the culture condition (50 degrees C) EG and CBH maintained 59% and 68% activity, which showed heat and alkali resistant characteristics.
Cell Culture Techniques ; Cellulase ; biosynthesis ; isolation & purification ; metabolism ; Cellulose ; metabolism ; Glucan 1,4-beta-Glucosidase ; biosynthesis ; metabolism ; Oryza ; Penicillium ; cytology ; enzymology ; isolation & purification ; Plant Stems ; microbiology ; Temperature ; beta-Glucosidase ; biosynthesis ; metabolism

A phage display single-chain variable fragment (scFv) library against Bursaphelenchus xylophilus cellulase (BXC) was constructed and used to screen the specific antibodies binding to BXC. The total RNA was extracted from fresh spleens of BALB/C mice immunized with BXC. Gene fragments encoding VH and VL were amplified by RT-PCR and assembled into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The recombinant fragments were cloned into the phagemids (pCANTABSE) and electroporated into E. coli TG1. The recombinant phagemids were rescued by reinfection of helper phage M13K07. The repertoire of the phage display antibody was about 5 x 10(4). The specific antibodies against BXC were obtained after five rounds of affinity selection. The positive phage clone was used to infect E. coli HB2151. SDS-PAGE and western blot analysis showed that the soluble scFv antibodies expressed bound specifically to BXC. The studies laid foundation for quarantine and pathological study of Bursaphelenchus xylophilu.
Animals ; Cellulase ; genetics ; immunology ; Cloning, Molecular ; Electroporation ; Escherichia coli ; genetics ; metabolism ; Female ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; Mice ; Mice, Inbred BALB C ; Nematoda ; enzymology ; Peptide Library ; Pinus ; parasitology ; Plant Diseases ; parasitology ; Recombinant Proteins ; biosynthesis ; genetics