Immobilization of malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103: properties and application in starch hydrolysis

BACKGROUND: A malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103 immobilized in calcium alginate beads was repeatedly used in batch processes of starch hydrolysis. The degree of starch degradation and operational stability of the immobilized system were optimized by varying the physical characteristics and composition of the beads. The products formed from hydrolysis of various starches by α‐amylase immobilized in different supports were analyzed. RESULTS: Immobilized beads prepared from 3% (w/v) alginate and 4% (w/v) CaCl₂ were suitable for up to 10 repeated uses, losing only 25% of their efficiency. On addition of 1% silica gel to alginate prior to gelation, the operational stability of the immobilized enzyme was enhanced to 20 cycles of operation, retaining > 90% of the initial efficiency. Distribution of malto‐oligosaccharides in the starch hydrolyzate depended on the type of starch, reaction time and mode of immobilization. Soluble starch and potato starch formed a wide range of malto‐oligosaccharides (G1–G5). Starches from wheat, rice and corn formed a narrow range of smaller oligosaccharides (G1–G3) as the major products. CONCLUSION: The immobilized beads of α‐amylase from KCC103 prepared from alginate plus silica gel showed high efficiency and operational stability for hydrolysis of starch. This immobilized system is useful for production of malto‐oligosaccharides applied in the food and pharmaceutical industries. Since this KCC103 amylase can be produced at low cost utilizing agro‐residues in a short time and immobilized enzyme can be recycled, the overall cost of malto‐oligosaccharide production would be economical for industrial application. Copyright © 2008 Society of Chemical Industry     

Preparation of chitosan‐coated magnetite nanoparticles and application for immobilization of laccase

In this study, immobilization of laccase (L) enzyme on magnetite (Fe₃O₄) nanoparticles was achieved, so that the immobilized enzyme could be used repeatedly. For this purpose, Fe₃O₄ nanoparticles were coated and functionalized with chitosan (CS) and laccase from Trametes versicolor was immobilized onto chitosan‐coated magnetic nanoparticles (Fe₃O₄‐CS) by adsorption or covalent binding after activating the hydroxyl groups of chitosan with carbodiimide (EDAC) or cyanuric chloride (CC). For chitosan‐coated magnetic nanoparticles, the thickness of CS layer was estimated as 1.0–4.8 nm by TEM, isoelectric point was detected as 6.86 by zeta (ζ)‐potential measurements, and the saturation magnetization was determined as 25.2 emu g^?1 by VSM, indicating that these nanoparticles were almost superparamagnetic. For free laccase and immobilized laccase systems, the optimum pH, temperature, and kinetic parameters were investigated; and the change of the activity against repeated use of the immobilized systems were examined. The results indicated that all immobilized systems retained more than 71% of their initial activity at the end of 30 batch uses. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Chitosaneous hydrogel beads for immobilizing neutral protease for application in the preparation of low molecular weight chitosan and chito‐oligomers

Enzyme hydrolysis with immobilized neutral protease was carried out to produce low molecular weight chitosan (LMWC) and chito‐oligomers. Neutral protease was immobilized on (CS), carboxymethyl chitosan (CMCS), and N‐succinyl chitosan (NSCS) hydrogel beads. The properties of free and immobilized neutral proteases on chitosaneous hydrogel beads were investigated and compared. Immobilization enhanced enzyme stability against changes in pH and temperature. When the three different enzyme supports were compared, the neutral protease immobilized on CS hydrogel beads had the highest thermal stability and storage stability, and the enzyme immobilized on NSCS hydrogel beads had the highest activity compared to those immobilized on the other supports, despite its lower protein loading. Immobilized neutral protease on all the three supports had a higher K_m (Michaelis‐Menten constant) than free enzyme. The V_max (maximum reaction velocity) value of neutral protease immobilized on CS hydrogel beads was lower than the free enzyme, whereas the V_max values of enzyme immobilized on CMCS and NSCS hydrogel beads were higher than that of the free enzyme. Immobilized neutral protease on CS, CMCS, and NSCS hydrogel beads retained 70.4, 78.2, and 82.5% of its initial activity after 10 batch hydrolytic cycles. The activation energy decreased for the immobilization of neutral protease on chitosaneous hydrogel beads. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3743–3750, 2006     

Synthesis and characterization of PMMA composites activated with starch for immobilization of L‐asparaginase

Poly (methyl methacrylate) (PMMA)–starch composites were prepared by emulsion polymerization technique for L‐asparaginase (L‐ASNase) immobilization as highly activated support. The hydroxide groups on the prepared composites offer a very simple, mild and firm combination for enzyme immobilization. The pure PMMA and PMMA‐starch composites were characterized as structural, thermal and morphological. PMMA‐starch composites were found to have better thermal stability and more hydrophilic character than pure PMMA. L‐ASNase was immobilized onto PMMA‐starch composites contained the different ratio of starch (1, 3, 5, and 10 wt %). Immobilized L‐ASNase showed better performance as compared to the native enzyme in terms of thermal stability and pH. K_m value of immobilized enzyme decreased approximately eightfold compared with the native enzyme. In addition to, immobilized L‐ASNase was found to retain 60% of activity after 1‐month storage period at 4 °C. Therefore, PMMA‐starch composites can be provided more advantageous in terms of enzymatic affinity, thermal, pH and storage stability as L‐ASNase immobilization matrix. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43421.     

Covalent immobilization of naringinase for the transformation of a flavonoid

Naringinase (EC 3.2.1.40) from Penicillium sp was immobilized by covalent binding to woodchips to improve its catalytic activity. The immobilization of naringinase on glutaraldehyde‐coated woodchips (600 mg woodchips, 10 U naringinase, 45 °C, pH 4.0 and 12h) through 1% glutaraldehyde cross‐linking was optimized. The pH–activity curve of the immobilized enzyme shifted toward a lower pH compared with that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of the enzyme. The immobilized naringinase was stable during storage at 4 °C. No loss of activity was observed when the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency of immobilization was 120%, while soluble naringinase afforded 82% efficacy for the hydrolysis of standard naringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76% debittering efficiency. Copyright © 2005 Society of Chemical Industry     

Synthesis and characterization of crosslinked chitosan immobilized on bentonite and its grafted products with polyaniline

Chitosan immobilized bentonite (CIB) namely chitosan‐coated bentonite (5% chitosan content) was synthesized in 2% acetic acid solution, followed by crosslinking, using epichlorohydrien (ECH). The so‐obtained crosslinked chitosan immobilized on bentonite (CIB‐ECH) and CIB composites were grafted with polyaniline (PANI) through oxidative‐radical copolymerization using ammonium peroxydisulfate in acidic medium to produce PANI‐grafted crosslinked chitosan immobilized on bentonite (PANI‐g‐CIB‐ECH) and PANI‐grafted‐chitosan immobilized on bentonite (PANI‐g‐CIB) composites, respectively. The resultant composites were characterized by using X‐ray diffraction (XRD), thermo gravimetric analysis/differential scanning calorimetry, scanning electron microscopy, Fourier transform infrared (FTIR), and electrical conductivity. XRD and FTIR analyses indicate that chitosan was not intercalated into the silicate layer. Also the electrical conductivity elucidates that the grafted composites fall in the range required for the application as electrostatic dissipation. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41078.     

Immobilization of chitosan on nylon 6,6 and pet granules through hydrolysis pretreatment

Chitosan has been increasingly studied as an adsorbent for removing heavy metal ions and organic compounds from aqueous solutions. Most of the studies used chitosan in the form of flakes, powder, or hydrogel beads. This research investigates the immobilization of chitosan on other granular materials to overcome the poor mechanical property of chitosan and offers the potential for chitosan to be used as a regenerable adsorbent. Nylon 6,6 and poly(ethylene terephthalate) (PET) granules were partially hydrolyzed under an acidic or alkaline condition to allow chitosan to be coated or immobilized on the granules' surfaces. The surface morphologies of nylon 6,6 or PET granules before and after hydrolysis and those with immobilized chitosan layer were examined by scanning electron microscopy (SEM), and their surface properties were characterized through ζ‐potential analysis and X‐ray photoelectron spectroscopy. The immobilization of chitosan on nylon 6,6 or PET granules was identified to be through the formation of the salt structure (–NH…^?OOC–) between the surfaces of hydrolyzed nylon 6,6 or PET granules and the chitosan layer. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3973–3979, 2003     

Hydrolysis of Penicillin G by Penicillin G Acylase Immobilized on Chitosan Microbeads in Different Reactor Systems

The kinetic parameters for penicillin G hydrolysis in systems with penicillin G acylase from Escherichia coli (free and immobilized on activated chitosan microbeads produced by electrostatic extrusion) were determined. The obtained kinetic results indicated that both systems (free and immobilized) are inhibited by high concentrations of the substrate (penicillin G) as well as by products of the reaction (6‐aminopenicillanic acid and phenylacetic acid). The microbeads appeared convenient for penicillin G acylase immobilization reducing negative inhibitory effects. The hydrolysis was also investigated in a packed bed reactor. The derived kinetic model predicted good hydrolysis rates in the reactor while the system with recirculation of the reaction mixture proved to be a potentially favorable solution providing operation at low shear stresses and possibly higher hydrolysis rates than in the packed bed reactor alone.     

Influence of chitosan derivatization on its physicochemical characteristics and its use as enzyme support

Chitosan was derivatized by two methodologies for analyzing their effect on chitosan physicochemical characteristics and its applicability as carrier for Bacillus circulans β‐galactosidase immobilization. Glutaraldehyde (GA) and epichlorohydrin (EPI) were used for crosslinking and activation of chitosan, producing the corresponding supports (C‐GA and C‐EPI‐EPI) after a one‐step and a two‐step process, respectively. The spherical shape and mean diameter of chitosan particles was not significantly affected by polymer derivatization, while Fourier transform infrared analysis showed that in both cases, chitosan polymer was chemically modified. TGA analysis indicated that C‐EPI‐EPI was the most thermally stable. The high degree of activation of C‐EPI‐EPI (586 μmol of aldehydes/g) resulted in the highest loss of activity during immobilization; hence a support with 100 μmol of aldehydes/g was produced (C‐EPI‐EPI₁₀₀). The highest expressed activity (89.3 IU/g) was obtained with the enzyme immobilized in C‐GA, while the biocatalyst with highest thermal stability at 60°C was obtained with C‐EPI‐EPI₁₀₀ (half‐life was 84‐fold higher than the one of the soluble enzyme). The best compromise between biocatalyst expressed activity and thermal stability corresponded to β‐galactosidase immobilized in C‐EPI‐EPI₁₀₀. According to this study, chitosan derivatized with EPI is a thermally stable carrier appropriate for producing highly stable immobilized B. circulans β‐galactosidase. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40171.     

Immobilization of lipase on hydrogels: Structural aspects of polymeric matrices as determinants of enzyme activity in different physical environments

Well‐defined and characterized polymeric matrices showing close chemical similarities but wide differences in water uptake and swellability in aqueous medium were used for lipase immobilization. Biphasic networks of 2‐hydroxypropylcellulose (HPC) were synthesized with acrylamide (AAm), methacrylamide (MAAm), N‐isopropylacrylamide (N‐i‐PAAm), and 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid (AMPSA) and simultaneously crosslinked with N,N‐methylene bisacrylamide in aqueous medium by using simultaneous γ‐radiation technique. Lipase enzyme was produced from a mesophilic bacterial isolate (HBK‐8) and was immobilized onto all the matrices by adsorption method. The activity of the immobilized enzyme was optimized for pH, temperature, and amount of crude enzyme and effect of dehydration. High relative activity for the immobilized enzymes was observed and loss of activity with time was minimal; reusability was found to be good. The activity of the immobilized enzyme was also observed to be good in both esterification and hydrolysis of esters. In the present study, lipase immobilization, hydrolysis of p‐nitrophenyl palmitate, and optimum pH and temperature for substrate hydrolysis were evaluated for different matrices to study polymer structure and enzyme activity relationship in diverse physical environments. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3135–3143, 2004     

UV‐cured natural polymer‐based membrane for biosensor application

Chitosan, a natural product, is inherently biodegradable, biocompatible, and nontoxic. These properties make chitosan ideal for inclusion in matrices designed for use in enzyme immobilization for clinical analysis. This study demonstrates the feasibility of using chitosan in electrochemical biosensor fabrication. The enzyme sulfite oxidase (SOX) was covalently immobilized onto the matrix of chitosan–poly(hydroxyethyl methacrylate) (chitosan–pHEMA), a natural/synthetic polymer hybrid obtainable via UV curing. p‐Benzoquinone, which served as an electron transfer mediator, was coupled onto the polymer network for activation of the chitosan–pHEMA copolymer, after completion of the photo‐induced polymerization reaction. The biological activity of the immobilized SOX and the electroactivity of the coupled p‐benzoquinone were investigated. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 79: 466–472, 2001     

Effect of immobilized neutral protease on the preparation and physicochemical properties of low molecular weight chitosan and chito‐oligomers

Neutral protease was immobilized on glutaraldehyde‐pretreated N‐succinyl chitosan hydrogel beads and the biocatalyst obtained was used for the preparation of low molecular weight chitosan and chito‐oligomers with molecular weight of 1.9–23.5 kDa from commercial chitosan. Factors affecting the chitinolytic hydrolysis were described. The degradation was monitored by gel permeation chromatography. The structure of degraded chitosan was characterized by Fourier transform infrared, X‐ray diffraction and liquid chromatography‐mass spectrometry. Immobilized neutral protease showed optimal depolymerization at pH 5.7 and 50°C. The degree of deacetylation of the hydrolysates did not change compared to that of the initial chitosan. The decrease of molecular weight led to transformation of crystal structure but the chemical structures of residues were not modified. The degree of polymerization of chito‐oligomers was mainly from 3 to 8. The method allows cyclic procedures of immobilized enzyme and N‐succinyl chitosan support utilization, and is suitable for a large‐scale production of the low molecular weight chitosan and chito‐oligomers free of protein admixtures. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102:4185–4193, 2006     

Poly(2‐hydroxyethylmethacrylate)/chitosan dye and different metal‐ion‐immobilized interpenetrating network membranes: Preparation and application in metal affinity chromatography

Composite membranes were synthesized with 2‐hydroxyethylmethacrylate and chitosan (pHEMA/chitosan) via an ultraviolet‐initiated photopolymerization technique in the presence of an initiator (α,α′‐azobisisobutyronitrile). The interpenetrating network (IPN) membranes were improved by the immobilization of dye molecules via hydroxyl and amino groups on the membrane surfaces from the IPNs. A triazidine dye (Procion Green H‐4G) was covalently immobilized as a ligand onto the IPN membranes. The protein showed various affinities to different chelated metal ions on the membrane surfaces that best matched its own distribution of functional sites, resulting in a distribution of binding energies. In support of this interpretation, two different metal ions, Zn(II) and Fe(III), were chelated with the immobilized dye molecules. The adsorption and binding characteristics of the different metal‐ion‐chelated dye‐immobilized IPN membranes for the lysozyme were investigated with aqueous solutions in magnetically stirred cells. The experimental data were analyzed with two adsorption kinetic models, pseudo‐first‐order and pseudo‐second‐order, to determine the best fit equation for the adsorption of lysozyme onto IPN membranes. The second‐order equation for the lysozyme–dye–metal‐chelated IPN membrane systems was the most appropriate equation for predicting the adsorption capacity for all the tested adsorbents. The reversible lysozyme adsorption on the dye‐immobilized and metal‐ion‐chelated membranes obeyed the Temkin isotherm. The lysozyme adsorption capacity of the pHEMA/chitosan dye, pHEMA/chitosan dye–Zn(II), and pHEMA/chitosan dye–Fe(III) membranes were 2.54, 2.85, and 3.64 mg cm^?2, respectively. The nonspecific adsorption of the lysozyme on the plain pHEMA/chitosan membrane was about 0.18 mg cm^?2. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1843–1853, 2003     

Effect of degree of deacetylation on solubility of low‐molecular‐weight chitosan produced via enzymatic breakdown of chitosan

Chitosan has emerged as a unique biomaterial, possessing scope in diverse applications in the biomedical, food and chemical industries. However, its high molecular weight is a concern when handling the polymer. Various techniques have been explored for depolymerization of this polymer, wherein enzymes have emerged as the most economic method having minimum degrading effect on the polymer and resulting in formation of side products. Chitosan can be depolymerized using a broad range of enzymes. In this study, various enzymes like α‐amylase, papain, pepsin and bromelain were employed to depolymerize chitosan and convert it into its lower molecular weight counterpart. Further, attempts were made to elucidate the process of depolymerization of chitosan, primarily by determining the change in its viscosity and hence its molecular weight. The process of depolymerization was optimized using a one‐factor‐at‐a‐time approach. The molecular weight of the resultant chitosan was estimated using gel permeation chromatography and infrared spectroscopy. These studies revealed a considerable decrease in molecular weights of chitosan depolymerized by pepsin, papain, bromelain and α‐amylase, resulting in recovery of the low‐molecular‐weight chitosan of 76.09 ± 5, 74.18 ± 5, 55.75 ± 5 and 49.18 ± 5%, respectively. Maximum yield and depolymerization were obtained using pepsin and papain due to their enzymatic recognition pattern, which was also validated using studies involving molecular dynamics. © 2019 Society of Chemical Industry     

Removal of alkylphenols by the combined use of tyrosinase immobilized on ion‐exchange resins and chitosan beads

Mushroom tyrosinase was covalently immobilized on a poly(acrylic acid)‐type, weakly acidic cation‐exchange resin (Daiaion WK10, Mitsubishi Chemical Corp., Tokyo, Japan) with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride salt as a water‐soluble carbodiimide. Ion‐exchange resins immobilized with tyrosinase were packed in one column, and crosslinked chitosan beads prepared with epichlorohydrin were packed in another column. The enzymatic activity was modified by covalent immobilization, and the immobilized tyrosinase had a high activity in the temperature range of 30–45°C and in the pH range of 7–10. When solutions of various alkylphenols were circulated through the two columns packed with tyrosinase‐immobilized ion‐exchange resins and crosslinked chitosan beads at 45°C and pH 7 (the optimum conditions determined for p‐cresol), alkylphenols were effectively removed through quinone oxidation with immobilized tyrosinase and subsequent quinone adsorption on chitosan beads. The use of chemically crosslinked chitosan beads in place of commercially available chitosan beads was effective in removing alkylphenols from aqueous solutions in shorter treatment times. The removal efficiency increased with an increase in the amount of crosslinked chitosan beads packed in the column because the rate of quinone adsorption became higher than the rate of enzymatic quinone generation. The activity of tyrosinase was iteratively used by covalent immobilization on ion‐exchange resins. One of the most important findings obtained in this study is the fact that linear and branched alkylphenols suspected of weak endocrine‐disrupting effects were effectively removed from aqueous solutions. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Immobilization of α-amylase on zirconia: A heterogeneous biocatalyst for starch hydrolysis

α-Amylase was immobilized on zirconia via adsorption. The support and the immobilized enzymes were characterized using XRD, IR spectra and N₂ adsorption studies. The efficiency of immobilized enzymes for starch hydrolysis was tested in a batch reactor. The effect of calcination temperatures on properties of the support as well as upon immobilization was studied. From XRD, IR and N₂ adsorption studies it was confirmed that the enzyme was adsorbed on the external surface of the support. pH, buffer concentration and substrate concentration had a significant influence on the activity of immobilized enzyme. Immobilization improved the pH stability of the enzyme. The Michaelis–Menten kinetic constants were calculated from Hanes–Woolf plot. K_m for immobilized systems was higher than the free enzyme indicating a decreased affinity by the enzyme for its substrate, which may be due to interparticle diffusional mass transfer restrictions.     

Integration of flocculation and adsorptive immobilization of Pseudomonas delafieldii R‐8 for diesel oil biodesulfurization

BACKGROUND: Deep desulfurization of hydrocarbon fuels is receiving increasing worldwide attention because of the increasingly stringent regulations to meet the requirement of environmental protection. Biodesulfurization (BDS) is being explored as either an alternative or complementary process to the conventional oil refining technologies. The whole cell immobilization technique is of great importance for accelerating the industrialization of BDS. An effective technique for a BDS process employing flocculation and integration with immobilization was developed. RESULTS: Pseudomonas delafieldii R‐8 cells were successfully flocculated and immobilized by directly adding chitosan and celite into the culture broth. The one‐step immobilized R‐8 cells exhibited good catalytic activity and retained at least 85% activity after six cycles of repeated‐batch desulfurization. Extensive biodesulfurization of diesel oil resulted in 82% reduction of total sulfur from 123 to 22 µg g^?1 in 24 h. CONCLUSIONS: A novel and simple technique was developed using chitosan flocculation and integration with cell immobilization onto celite for dibenzothiophene BDS. The present report indicates that integration of flocculation and immobilization may provide a continuous and efficient method of BDS. Copyright © 2010 Society of Chemical Industry     

Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N₂ adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. ²⁷Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N₂ adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. K _m was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.     

Chitosan‐treated polypropylene matrix as immobilization support for lactic acid production using Lactobacillus plantarum NCIM 2084

Adsorption coupled with electrostatic interaction as an immobilization technique is an important microbial cell immobilization technique. Treatment of the polymer matrix with the cationic surface treating agent chitosan for lactic acid production has been studied. Cells of Lactobacillus plantarum NCIM 2084 were immobilized on a polypropylene (PP) matrix treated with different concentrations of chitosan. The biocatalyst adsorbed on the 1.0 g dm^?3 chitosan‐treated PP matrix proved to be most effective. Repeated batch fermentation experiments showed that the immobilized biocatalyst could be recycled effectively 11 times. Studies were also carried out in a packed bed reactor with media recirculation. A high productivity of 7.66 g dm^?3 h^?1 could be obtained with a conversion of 94% and a yield of 97% at an average residence time of 30 h. © 2001 Society of Chemical Industry     

One‐step production of 2,3‐butanediol from starch by secretory over‐expression of amylase in Klebsiella pneumoniae

BACKGROUND: 2,3‐Butanediol (2,3‐BD) is a valuable chemical that can be biosynthesized from many kinds of substrates. For commercial biological production of 2,3‐BD, it is desirable to use cheap substrate without pretreatment, such as starch. However, there have been few reports on the production of 2,3‐BD directly from starch. RESULTS: In this work, gene malS coding for α‐amylase (EC 3.2.1.1) precursor was inserted into plasmid pUC18K, and secretory over‐expression of α‐amylase was achieved by engineered Klebsiella pneumoniae. The extracellular recombinant amylase accelerated the hydrolyzation of starch, and one‐step production of 2,3‐BD from starch was carried out by engineered K. pneumoniae. A 2,3‐BD concentration of 3.8 g L^?1 and yield of 0.19 g 2,3‐BD g^?1 starch were obtained after 24 h fermentation. CONCLUSION: The one‐step production of 2,3‐BD from starch was achieved by secretory over‐expression of amylase in K. pneumoniae. This would simplify the process and reduce the production cost considerably by enabling use of starch with minimal pretreatment. Copyright © 2008 Society of Chemical Industry