Badal C. Saha

α-L-Arabinofuranosidases: biochemistry, molecular biology and application in biotechnology.

Interest in the alpha-L-arabinofuranosidases has increased in recent years because of their application in the conversion of various hemicellulosic substrates to fermentable sugars for subsequent production of fuel alcohol. Xylanases, in conjunction with alpha-L-arabinofuranosidases and other accessory enzymes, act synergistically to degrade xylan to component sugars. The induction of alpha-L-arabinofuranosidase production, physico-chemical characteristics, substrate specificity, and molecular biology of the enzyme are described. The current state of research and development of the arabinofuranosidases and their role in biotechnology are presented.

Dilute acid pretreatment, enzymatic saccharification, and fermentation of rice hulls to ethanol.

Rice hulls, a complex lignocellulosic material with high lignin (15.38 ± 0.2%) and ash (18.71 ± 0.01%) content, contain 35.62 ± 0.12% cellulose and 11.96 ± 0.73% hemicellulose and has the potential to serve as a low‐cost feedstock for production of ethanol. Dilute H2SO4 pretreatments at varied temperature (120–190 °C) and enzymatic saccharification (45 °C, pH 5.0) were evaluated for conversion of rice hull cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from rice hulls (15%, w/v) by dilute H2SO4 (1.0%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β‐glucosidase, xylanase, esterase, and Tween 20 was 287 ± 3 mg/g (60% yield based on total carbohydrate content). Under this condition, no furfural and hydroxymethyl furfural were produced. The yield of ethanol per L by the mixed sugar utilizing recombinant Escherichia coli strain FBR 5 from rice hull hydrolyzate containing 43.6 ± 3.0 g fermentable sugars (glucose, 18.2 ± 1.4 g; xylose, 21.4 ± 1.1 g; arabinose, 2.4 ± 0.3 g; galactose, 1.6 ± 0.2 g) was 18.7 ± 0.6 g (0.43 ± 0.02 g/g sugars obtained; 0.13 ± 0.01 g/g rice hulls) at pH 6.5 and 35 °C. Detoxification of the acid‐ and enzyme‐treated rice hull hydrolyzate by overliming (pH 10.5, 90 °C, 30 min) reduced the time required for maximum ethanol production (17 ± 0.2 g from 42.0 ± 0.7 g sugars per L) by the E. coli strain from 64 to 39 h in the case of separate hydrolysis and fermentation and increased the maximum ethanol yield (per L) from 7.1 ± 2.3 g in 140 h to 9.1 ± 0.7 g in 112 h in the case of simultaneous saccharification and fermentation.

Ethanol Production from Alkaline Peroxide Pretreated Enzymatically Saccharified Wheat Straw

Wheat straw used in this study contained 44.24 ± 0.28% cellulose and 25.23 ± 0.11% hemicellulose. Alkaline H2O2 pretreatment and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from wheat straw (8.6%, w/v) by alkaline peroxide pretreatment (2.15% H2O2, v/v; pH 11.5; 35 °C; 24 h) and enzymatic saccharification (45 °C, pH 5.0, 120 h) by three commercial enzyme preparations (cellulase, β‐glucosidase, and xylanase) using 0.16 mL of each enzyme preparation per g of straw was 672 ± 4 mg/g (96.7% yield). During the pretreatment, no measurable quantities of furfural and hydroxymethyl furfural were produced. The concentration of ethanol (per L) from alkaline peroxide pretreated enzyme saccharified wheat straw (66.0 g) hydrolyzate by recombinant Escherichia coli strain FBR5 at pH 6.5 and 37 °C in 48 h was 18.9 ± 0.9 g with a yield of 0.46 g per g of available sugars (0.29 g/g straw). The ethanol concentration (per L) was 15.1 ± 0.1 g with a yield of 0.23 g/g of straw in the case of simultaneous saccharification and fermentation by the E. coli strain at pH 6.0 and 37 °C in 48 h.

Pretreatment and enzymatic saccharification of corn fiber

Corn fiber consists of about 20% starch, 14% cellulose, and 35% hemicellulose, and has the potential to serve as a low-cost feedstock for production of fuel ethanol. Several pretreatments (hot water, alkali, and dilute, acid) and enzymatic saccharification procedures were evaluated for the conversion of corn fiber starch, cellulose, and hemicellulose to monomeric sugars. Hot water pretreatment (121°C, 1 h) facilitated the enzymatic sacch arification of starch and cellulose but not hemicellulose. Hydrolysis of corn fiber pretreated with alkali un dersimilar conditions by enzymatic means gave similar results. Hemicellulose and starch components were converted to monomeric sugars by dilute H2SO4 pretreatment (0.5–1.0%, v/v) at 121°C. Based on these findings, a method for pretreatment and enzymatic saccharification of corn fiber is presented. It in volves the pretreatment of corn fiber (15% solid, w/v) with dilute acid (0.5% H2SO4, v/v) at 121°C for 1 h, neutralization to pH 5.0, then saccharification of the pretreated corn fiber material with commercial cellulase and β-glucosidase preparations The yield of monomeric sugars from corn fiber was typically 85–100% of the theoretical yield.

Butanol Production from Corn Fiber Xylan Using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L·h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L·h). CFAX was hydrolyzed by xylan‐hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.

Debittering of protein hydrolyzates.

Enzymatic hydrolysis of proteins frequently results in bitter taste, which is due to the formation of low molecular weight peptides composed of mainly hydrophobic amino acids. Methods for debittering of protein hydrolyzates include selective separation such as treatment with activated carbon, extraction with alcohol, isoelectric precipitation, chromatography on silica gel, hydrophobic interaction chromatography, and masking of bitter taste. Bio-based methods include further hydrolysis of bitter peptides with enzymes such as aminopeptidase, alkaline/neutral protease and carboxypeptidase, condensation reactions of bitter peptides using protease, and use of Lactobacillus as a debittering starter adjunct. The causes for the production of bitter peptides in various food protein hydrolyzates and the development of methods for the prevention, reduction, and elimination of bitterness as well as masking of bitter taste in enzymatic protein hydrolyzates are presented.

Comparison of pretreatment strategies for enzymatic saccharification and fermentation of barley straw to ethanol

Barley straw used in this study contained 34.3% cellulose, 23.0% hemicellulose and 13.3% lignin (moisture, 6.5%). Several pretreatments (dilute acid, lime and alkaline peroxide) and enzymatic saccharification procedures were evaluated for the conversion of barley straw to monomeric sugars. The maximum release of sugars (glucose, 384 mg; xylose, 187 mg; arabinose, 32 mg; total sugars, 604 mg/g; 94% of maximum theoretical sugar yield) from barley straw (10%, w/v) was obtained by alkaline peroxide (2.5% H(2)O(2), pH 11.5) pretreatment (35 degrees C, 24 hours) and enzymatic saccharification (45 degrees C, pH 5.0, 120 hours) after diluting 2 times before adding a cocktail of three commercial enzyme preparations (cellulase, beta-glucosidase and hemicellulase) each at the dose level of 0.15 ml/g of straw. Dilute acid and lime pretreatments followed by enzymatic saccharification generated 566 mg (88% yield) and 582 mg (91% yield) total sugars/g of barley straw, respectively. The yield of ethanol from the dilute acid pretreated and enzymatically saccharified barley straw hydrolyzate (23.7g sugars/L) was 11.4g/L (0.48g/g available sugars, 0.26g/g straw) by the mixed sugar utilizing recombinant Escherichia coli strain FBR5 in 17 hours. The ethanol yields were 11.4 and 11.9g/L from 24.4 and 26.2g sugars/L obtained from lime and alkaline peroxide pretreated barley straw, respectively. No inhibition of fermentation occurred by any of the three pretreatments under the conditions used.

Purification and properties of an extracellular β-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.

Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production.

Dilute H(3)PO(4) (0.0-2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pretreatment conditions were optimized for temperature, acid loading, and time using central composite design. Optimal pretreatment conditions were chosen to promote sugar yields following enzymatic digestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maximum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180°C for 15min while highest yield for xylose (91.4%) was observed from corn stover pretreated with 1% (v/v) acid at 160°C for 10min. About 26.4±0.1g ethanol was produced per L by recombinant Escherichia coli strain FBR5 from 55.1±1.0g sugars generated from enzymatically hydrolyzed corn stover (10%, w/w) pretreated under a balanced optimized condition (161.81°C, 0.78% acid, 9.78min) where only 0.4±0.0g furfural and 0.1±0.0 hydroxylmethyl furfural were produced.

Process integration for simultaneous saccharification, fermentation, and recovery (SSFR): Production of butanol from corn stover using Clostridium beijerinckii P260

A simultaneous saccharification, fermentation, and recovery (SSFR) process was developed for the production of acetone-butanol-ethanol (AB or ABE), of which butanol is the main product, from corn stover employing Clostridium beijerinckii P260. Of the 86 g L(-1) corn stover provided, over 97% of the sugars were released during hydrolysis and these were fermented completely with an ABE productivity of 0.34 g L(-1)h(-1) and yield of 0.39. This productivity is higher than 0.31 g L(-1)h(-1) when using glucose as a substrate demonstrating that AB could be produced efficiently from lignocellulosic biomass. Acetic acid that was released from the biomass during pretreatment and hydrolysis was also used by the culture to produce AB. An average rate of generation of sugars during corn stover hydrolysis was 0.98 g L(-1)h(-1). In this system AB was recovered using vacuum, and as a result of this (simultaneous product recovery), 100% sugars were used by the culture.