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Y.-h. Percival Zhang - One of the best experts on this subject based on the ideXlab platform.

  • Recyclable cellulose-containing magnetic nanoparticles: immobilization of Cellulose-Binding Module-tagged proteins and a synthetic metabolon featuring substrate channeling.
    Journal of materials chemistry. B, 2013
    Co-Authors: Suwan Myung, Chun You, Y.-h. Percival Zhang
    Abstract:

    Easily recyclable cellulose-containing magnetic nanoparticles were developed for immobilizing family 3 Cellulose-Binding Module (CBM)-tagged enzymes/proteins and a self-assembled three-enzyme complex called the synthetic metabolon. Avicel (microcrystalline cellulose)-containing magnetic nanoparticles (A-MNPs) and two controls of dextran-containing magnetic nanoparticles (D-MNPs) and magnetic nanoparticles (MNPs) were prepared by a solvothermal method. Their adsorption ability was investigated by using CBM-tagged green fluorescence protein and phosphoglucose isomerase. A-MNPs had higher adsorption capacity and tighter binding on CBM-tagged proteins than the two control MNPs because of the high-affinity adsorption of CBM on cellulose. In addition, A-MNPs were used to purify and co-immobilize a three-enzyme metabolon through a CBM-tagged scaffoldin containing three different cohesins. The three-enzyme metabolon comprised of dockerin-containing triosephosphate isomerase, aldolase, and fructose 1,6-bisphosphatase was self-assembled because of the high-affinity interaction between cohesins and dockerins. Thanks to spatial organization of the three-enzyme metabolon on the surface of A-MNPs, the metabolon exhibited a 4.6 times higher initial reaction rate than the non-complexed three-enzyme mixture at the same enzyme loading. These results suggested that the cellulose-containing MNPs were new supports for immobilizing enzymes, which could be selectively recycled or removed from other biocatalysts by a magnetic force, and the use of enzymes immobilized on A-MNPs could be very useful to control the On/Off process in enzymatic cascade reactions.

  • Self-Assembly of Synthetic Metabolons through Synthetic Protein Scaffolds: One-Step Purification, Co-immobilization, and Substrate Channeling
    ACS synthetic biology, 2012
    Co-Authors: Chun You, Y.-h. Percival Zhang
    Abstract:

    One-step purification of a multi-enzyme complex was developed based on a mixture of cell extracts containing three dockerin-containing enzymes and one family 3 Cellulose-Binding Module (CBM3)-containing scaffoldin through high-affinity adsorption on low-cost solid regenerated amorphous cellulose (RAC). The three-enzyme complex, called synthetic metabolon, was self-assembled through the high-affinity interaction between the dockerin in each enzyme and three cohesins in the synthetic scaffoldin. The metabolons were either immobilized on the external surface of RAC or free when the scaffoldin contained an intein between the CBM3 and three cohesins. The immobilized and free metabolons containing triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase exhibited initial reaction rates 48 and 38 times, respectively, that of the non-complexed three-enzyme mixture at the same enzyme loading. Such reaction rate enhancements indicated strong substrate channeling among synthetic metabolons due to the clos...

  • Fusion of a family 9 Cellulose-Binding Module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose
    Applied Microbiology and Biotechnology, 2011
    Co-Authors: Zhiguang Zhu, Chenming Zhang, Y.-h. Percival Zhang
    Abstract:

    Clostridium thermocellum cellodextrin phosphorylase ( Ct CDP), a single-Module protein without an apparent carbohydrate-binding Module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, Ct CDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding Modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of Ct CDP. Fusion of any selected CBM with Ct CDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to Ct CDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. Ct CDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein Ct CDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).

  • Ultra-stable phosphoglucose isomerase through immobilization of Cellulose-Binding Module-tagged thermophilic enzyme on low-cost high-capacity cellulosic adsorbent.
    Biotechnology progress, 2011
    Co-Authors: Suwan Myung, Y.-h. Percival Zhang
    Abstract:

    One-step enzyme purification and immobilization were developed based on simple adsorption of a family 3 Cellulose-Binding Module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose (RAC). An open reading frame (ORF) Cthe0217 encoding a putative phosphoglucose isomerase (PGI, EC 5.3.1.9) from a thermophilic bacterium Clostridium thermocellum was cloned and the recombinant proteins with or without CBM were over-expressed in Escherichia coli. The rate constant (kcat) and Michaelis–Menten constant (Km) of CBM-free PGI at 60°C were 2,765 s−1 and 2.89 mM, respectively. PGI was stable at a high protein concentration of 0.1 g/L but deactivated rapidly at low concentrations. Immobilized CBM (iCBM)-PGI on RAC was extremely stable at ∼60°C, nearly independent of its mass concentration in bulk solution, because its local concentration on the solid support was constant. iCBM-PGI at a low concentration of 0.001 g/L had a half-life time of 190 h, approximately 80-fold of that of free PGI. Total turn-over number of iCBM-PGI was as high as 1.1 × 109 mole of product per mole of enzyme at 60°C. These results suggest that a combination of low-cost enzyme immobilization and thermoenzyme led to an ultra-stable enzyme building block suitable for cell-free synthetic pathway biotransformation that can implement complicated biochemical reactions in vitro. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.

  • Engineering of Clostridium phytofermentans Endoglucanase Cel5A for improved thermostability.
    Applied and environmental microbiology, 2010
    Co-Authors: Wenjin Liu, Zuoming Zhang, Y.-h. Percival Zhang
    Abstract:

    A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding Module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.

Peter Biely - One of the best experts on this subject based on the ideXlab platform.

  • purification and characterization of two forms of endo β 1 4 mannanase from a thermotolerant fungus aspergillus fumigatus imi 385708 formerly thermomyces lanuginosus imi 158749
    Biochimica et Biophysica Acta, 2004
    Co-Authors: Vladimir Puchart, Maria Vrsanska, Pavel Svoboda, Jan Pohl, Zumrut B Ogel, Peter Biely
    Abstract:

    Abstract Two extracellular endo-β-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with p I values in the range of 4.9–5.2 for MAN I and 4.75–4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 °C and were stable in the pH range 4.5–8.5 and up to 55 °C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1 H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both β-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a Cellulose-Binding Module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome ( http://www.sanger.ac.uk/Projects/A_fumigatus/ ).

  • purification and characterization of two forms of endo β 1 4 mannanase from a thermotolerant fungus aspergillus fumigatus imi 385708 formerly thermomyces lanuginosus imi 158749
    Biochimica et Biophysica Acta, 2004
    Co-Authors: Vladimir Puchart, Maria Vrsanska, Pavel Svoboda, Jan Pohl, Zumrut B Ogel, Peter Biely
    Abstract:

    Two extracellular endo-beta-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9-5.2 for MAN I and 4.75-4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 degrees C and were stable in the pH range 4.5-8.5 and up to 55 degrees C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both beta-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a Cellulose-Binding Module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome (http://sanger.ac.uk/Projects/A_fumigatus/).

David Wilson - One of the best experts on this subject based on the ideXlab platform.

  • Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization.
    Biotechnology and bioengineering, 2011
    Co-Authors: Maxim Kostylev, Jose M. Moran-mirabal, Larry P. Walker, David Wilson
    Abstract:

    Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Ther- mobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding Module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble sub- strate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate. These findings are relevant for kinetic modeling and microscopy studies of modular glycoside hydrolases. Biotechnol. Bioeng. 2012;109: 295-299. 2011 Wiley Periodicals, Inc.

  • Thermobifida fusca exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A.
    Systems and Synthetic Biology, 2010
    Co-Authors: Jonathan Caspi, Raphael Lamed, Diana C. Irwin, David Wilson, Yoav Barak, Rachel Haimovitz, Hadar Gilary, Edward A. Bayer
    Abstract:

    Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin Module and the cohesin Module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a Cellulose-Binding Module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action.

  • increased crystalline cellulose activity via combinations of amino acid changes in the family 9 catalytic domain and family 3c cellulose binding Module of thermobifida fusca cel9a
    Applied and Environmental Microbiology, 2010
    Co-Authors: Diana C. Irwin, David Wilson
    Abstract:

    Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding Module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.

  • Conversion of Thermobifida fusca free exoglucanases into cellulosomal components: Comparative impact on cellulose-degrading activity
    Journal of Biotechnology, 2008
    Co-Authors: Jonathan Caspi, Henri Pierre Fierobe, Raphael Lamed, Diana C. Irwin, David Wilson, Edward A. Bayer
    Abstract:

    Cellulosomes are multi-enzyme complexes produced by certain anaerobic bacteria that exhibit efficient degradation of plant cell wall polysaccharides. To understand their enhanced levels of hydrolysis, we are investigating the effects of converting a free-cellulase system into a cellulosomal one. To achieve this end, we are replacing the Cellulose-Binding Module of the native cellulases, produced by the aerobic bacterium Thermobifida fusca, with a cellulosome-derived dockerin Module of established specificity, to allow their incorporation into defined “designer cellulosomes”. In this communication, we have attached divergent dockerins to the two exoglucanases produced by T. fusca exoglucanase, Cel6B and Cel48A. The resultant fusion proteins were shown to bind efficiently and specifically to their matching cohesins, and their activities on several different cellulose substrates were compared. The lack of a Cellulose-Binding Module in Cel6B had a deleterious effect on its activity on crystalline substrates. In contrast, the dockerin-bearing family-48 exoglucanase showed increased levels of hydrolytic activity on carboxymethyl cellulose and on both crystalline substrates tested, compared to the wild-type enzyme. The marked difference in the response of the two exoglucanases to incorporation into a cellulosome, suggests that the family-48 cellulase is more appropriate than the family-6 enzyme as a designer cellulosome component.

Vladimir Puchart - One of the best experts on this subject based on the ideXlab platform.

  • purification and characterization of two forms of endo β 1 4 mannanase from a thermotolerant fungus aspergillus fumigatus imi 385708 formerly thermomyces lanuginosus imi 158749
    Biochimica et Biophysica Acta, 2004
    Co-Authors: Vladimir Puchart, Maria Vrsanska, Pavel Svoboda, Jan Pohl, Zumrut B Ogel, Peter Biely
    Abstract:

    Abstract Two extracellular endo-β-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with p I values in the range of 4.9–5.2 for MAN I and 4.75–4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 °C and were stable in the pH range 4.5–8.5 and up to 55 °C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1 H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both β-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a Cellulose-Binding Module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome ( http://www.sanger.ac.uk/Projects/A_fumigatus/ ).

  • purification and characterization of two forms of endo β 1 4 mannanase from a thermotolerant fungus aspergillus fumigatus imi 385708 formerly thermomyces lanuginosus imi 158749
    Biochimica et Biophysica Acta, 2004
    Co-Authors: Vladimir Puchart, Maria Vrsanska, Pavel Svoboda, Jan Pohl, Zumrut B Ogel, Peter Biely
    Abstract:

    Two extracellular endo-beta-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9-5.2 for MAN I and 4.75-4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 degrees C and were stable in the pH range 4.5-8.5 and up to 55 degrees C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both beta-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a Cellulose-Binding Module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome (http://sanger.ac.uk/Projects/A_fumigatus/).

Jiong Hong - One of the best experts on this subject based on the ideXlab platform.

  • A Simple Method for Beta-glucosidase Immobilization and Its Application in Soybean Isoflavone Glycosides Hydrolysis
    Biotechnology and Bioprocess Engineering, 2018
    Co-Authors: Hu Shenglin, Dongmei Wang, Jiong Hong
    Abstract:

    In this study, a simple, inexpensive and fast β-glucosidase immobilization system was constructed and evaluated in isoflavone glycosides hydrolysis. A β-glucosidase gene from Thermoascus aurantiacus IFO9748 was recombinantly expressed in Pichia pastoris KM71H and immobilized on regenerated amorphous cellulose (RAC) by fused cellulose binding Module 3. Through simple mixing cellulose and crude enzyme for 15 min under room temperature, 96.04% β-glucosidase was immobilized onto RAC. The optimum temperature for β-glucosidase activity was increased by 5oC after immobilization. The half-life (t½) of heat inactivation of immobilized enzyme at 60oC was improved over 8 folds. After 30 rounds recycled at 40oC, 96.9% daidzin and 98.9% genistin could still be hydrolyzed. A continuous hydrolysis system was also constructed, and at the flow rate of 0.2 mL/min after 30 h hydrolysis, 95.6% genistin and 90.2% daidzin can still be hydrolyzed. Combined the simple and high efficient enzyme immobilization procedure and inexpensive cellulose, this scalable and practical system may have broad prospects for industrial utilization.

  • Purification of a recombinant protein with Cellulose-Binding Module 3 as the affinity tag.
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Dongmei Wang, Jiong Hong
    Abstract:

    Easy-to-perform and low-cost protein purification methods are in high demand for the mass production of commonly used enzymes that play an important role in bioeconomy. A low-cost and rapid recombinant protein purification system was developed using CBM3 (family 3 Cellulose-Binding Module) as affinity tag. This protocol describes the purification of CBM3-fusion protein and tag-free protein expressed in Pichia pastoris using CBM3 as an affinity tag.

  • Expression of family 3 Cellulose-Binding Module (CBM3) as an affinity tag for recombinant proteins in yeast.
    Applied microbiology and biotechnology, 2011
    Co-Authors: Wen Wan, Dongmei Wang, Xiaolian Gao, Jiong Hong
    Abstract:

    Easy and low-cost protein purification methods for the mass production of commonly used enzymes that play important roles in biotechnology are in high demand. In this study, we developed a fast, low-cost recombinant protein purification system in the methylotrophic yeast Pichia pastoris using the family 3 Cellulose-Binding Module (CBM3)-based affinity tag. The codon of the cbm3 gene from Clostridium thermocellum was optimized based on the codon usage of P. pastoris. The CBM3 tag was then fused with enhanced green fluorescent protein (CBM3-EGFP) or with inulinase and expressed in P. pastoris to demonstrate its ability to function as an affinity tag in a yeast expression system. We also examined the effects of glycosylation on the secreted CBM3-tag. The secreted wild-type CBM3-EGFP was glycosylated; however, this had little influence on the adsorption of the fusion protein to the regenerated amorphous cellulose (RAC; maximum adsorption capacity of 319 mg/g). Two CBM3-EGFP mutants lacking glycosylation sites were also constructed. The three CBM3-EGFPs expressed in P. pastoris and the CBM3-EGFP expressed in Escherichia coli all had similar RAC adsorption capacity. To construct a tag-free recombinant protein purification system based on CBM3, a CBM3-intein-EGFP fusion protein was expressed in P. pastoris. This fusion protein was stably expressed and the self-cleavage of intein was efficiently induced by DTT or l-cysteine. In this study, we were able to purify the recombinant fusion protein with high efficiency using both intein and direct fusion-based strategies.

  • bioseparation of recombinant cellulose binding Module proteins by affinity adsorption on an ultra high capacity cellulosic adsorbent
    Analytica Chimica Acta, 2008
    Co-Authors: Jiong Hong, Xinhao Ye, Y Percival H Zhang, Yiran Wang
    Abstract:

    Abstract Low-cost protein purification methods are in high demand for mass production of low-selling price enzymes that play an important role in the upcoming bioeconomy. A simple protein purification method was developed based on affinity adsorption of a Cellulose-Binding Module-tagged protein on regenerated amorphous cellulose (RAC) followed by modest desorption. The biodegradable cellulosic adsorbent RAC had a very high protein-binding capacity of up to 365 mg of protein per gram of RAC. The specifically-bound CBM-protein on the external surface of RAC was eluted efficiently by ethyl glycol or glycerol. This protein separation method can be scaled up easily because it is based on simple solid/liquid unit operations. Five recombinant proteins (CBM-protein), regardless of intercellular or periplasmic form, were purified successfully for demonstration purpose.

  • simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self cleavage
    Journal of Chromatography A, 2008
    Co-Authors: Jiong Hong, Xinhao Ye, Y Percival H Zhang, Yiran Wang
    Abstract:

    A simple, low-cost, and scalable protein purification method was developed by using a biodegradable regenerated amorphous cellulose (RAC) with a binding capacity of up to 365 mg protein per gram of RAC. The recombinant protein with a Cellulose-Binding Module (CBM) tag can be specifically adsorbed by RAC. In order to avoid using costly protease and simplify purification process, a self-cleavage intein was introduced between CBM and target protein. The cleaved target protein can be liberated from the surface of RAC by intein self-cleavage occurring through a pH change from 8.0 to 6.5. Four recombinant proteins (green fluorescence protein, phosphoglucomutase, cellobiose phosphorylase, and glucan phosphorylase) have been purified successfully.