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Cellulases play a promising role in the bioconversion of renewable lignocellulosic biomass into fermentable sugars which are subsequently fermented to biofuels and other value-added chemicals. Besides biofuel industries, they are also in huge demand in textile, detergent, and paper and pulp industries. Low titres of cellulase production and processing are the main issues that contribute to high enzyme cost. The success of ethanol-based biorefinery depends on high production titres and the catalytic efficiency of cellulases functional at elevated temperatures with acid/alkali tolerance and the low cost. In view of their wider application in various industrial processes, stable cellulases that are active at elevated temperatures in the acidic-alkaline pH ranges, and organic solvents and salt tolerance would be useful. This review provides a recent update on the advances made in thermostable cellulases. Developments in their sources, characteristics and mechanisms are updated. Various methods such as rational design, directed evolution, synthetic & system biology and immobilization techniques adopted in evolving cellulases with ameliorated thermostability and characteristics are also discussed. The wide range of applications of thermostable cellulases in various industrial sectors is described., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The optimum values of the critical variables determined by the central composite design of response surface methodology (RSM) for maximum phytase production (1881.26 U g(-1) dry mouldy residue (DMR)) by Sporotrichum thermophile are 2.5 % Tween 80, 1.0 % yeast extract and 48 h of incubation period. Phytase production in the mixed substrate (sugarcane bagasse and wheat bran) fermentation enhanced 11.6-fold over the initial production as a consequence of optimization. Phytase titres are sustainable in flasks, trays and column bioreactor (1796 to 2095 U g(-1) DMR), thus validating the model and the process for large-scale phytase production. When the yeast extract was replaced with corn steep liquor (2 % w/v), a sustained enzyme titre (1890 U g(-1) DMR) was attained, making the process cost-effective. Among all the detergents, Tween 80 supported a higher phytase production than others. The enzyme efficiently liberated nutritional components from poultry feed (inorganic phosphate, soluble protein and reducing sugars) in a time-dependent manner.

Among matrices used for immobilizing Bacillus acidicola cells [calcium alginate, chitosan + alginate, scotch brite, and polyurethane foam (PUF)], α-amylase production was highest by PUF-immobilized cells (9.1 U ml(-1)), which is higher than free cells (7.2 U ml(-1)). The PUF-immobilized cells could be reused over seven cycles with sustained α-amylase production. When three variables (moisture, starch, and ammonium sulfate), which significantly affected enzyme production in solid-state fermentation (SSF), were optimized using response surface methodology, 5.6-fold enhancement in enzyme production was attained. The enzyme production in SSF is 3.8-fold higher than that in submerged fermentation. The bread made by supplementing dough with α-amylase of B. acidicola scored better than those with the xylanase of Bacillus halodurans and thermostable α-amylase of Geobacillus thermoleovorans.

The production of acidic α-amylase by a novel acidophilic bacterium Bacillus acidicola TSAS1 was optimized in submerged fermentation using statistical approaches. The process parameters that significantly affected α-amylase production (starch, K(2)HPO(4), inoculum size and temperature) were identified by Plackett and Burman design. The optimum levels of the significant variables as determined using central composite design of response surface methodology are starch (2.75%), K(2)HPO(4) (0.01%), inoculum size [2% (v/v) containing 1.9×10(8) CFU ml(-1)], and temperature (33°C). An overall 2.4 and 2.9-fold increase in enzyme production has been attained in batch and fed-batch fermentations in the laboratory fermentor, respectively., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Among physical and nutritional parameters optimized by "one variable at a time" approach, four cultural variables (sucrose, MgSO4 .7H2O, inoculum size, and incubation period) significantly affected glucoamylase production. These variables were, therefore, selected for optimization using response surface methodology. The p-values of the coefficients for linear effect of sucrose and inoculum size were less than 0.0001, suggesting them to be the key experimental variables in glucoamylase production. The enzyme production (34 U/ml) attained under optimized conditions (sucrose, 2%; MgSO4 .7H2O, 0.13%; yeast extract, 0.1%; inoculum size, 5 x 10(6) spores per 50 ml production medium; incubation time, 48 h; temperature, 40 degrees C; and pH 7.0) was comparable with the value predicted by polynomial model (34.2 U/ml). An over all 3.1-fold higher enzyme titers were attained due to response surface optimization. The experimental model was validated by carrying out glucoamylase production in shake flasks of increasing capacity (0.25-2.0 l) and 22-l laboratory bioreactors (stirred tank and airlift), where the enzyme production was sustainable. Furthermore, the fermentation time was reduced from 48 h in shake flasks to 32 h in bioreactors.

The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used animal feed ingredients tested to varying degrees in solid-state fermentation. Enzyme production increased to 180 U/g of dry moldy residue (DMR) in sesame oil cake at 120 h and 45 degrees C at the initial substrate-to-moisture ratio of 1:2.5 and aw of 0.95. Supplementation of sesame oil cake with glucose and ammonium sulfate further enhanced phytase titer (282 U/g of DMR). An overall 76% enhancement in phytase production was achieved owing to optimization. The mold secreted acid phosphatase, amylase, xylanase, and lipase along with phytase. By the action of phytase, inorganic phosphate was liberated efficiently, leading to dephytinization of sesame oil cake.

Abstract: The production of thermo-alkali-stable and cellulase-free xylanase of polyextremophilic novel Bacillus halodurans TSEV1 (MTCC 10962) was optimized in batch fermentation using statistical approaches. Plackett–Burman (PB) design identified temperature, inoculum size, KH2PO4 and wheat bran (WB) as the significant variables that affect xylanase production, and therefore, these variables have been optimized by Central composite design (CCD) matrix of response surface methodology (RSM). A 7.35-fold enhancement in xylanase production was attained due to optimization. The UV absorption spectrum of the compounds released by the enzyme action on unbleached pulp showed a characteristic peak at 280 nm indicating the presence of lignin related compounds in the filtrate. The materials released after biobleaching also displayed strong absorption at 237 and 465 nm. The enzyme dose 40 Ug−1, pH 10.0, and treatment time of 3 h are optimal for enzymatic pre-bleaching of wheat pulp that caused 14.6% reduction in kappa number and 5.6% enhancement in brightness of hand sheets without noticeable change in viscosity. This is the first report on xylanase production by a wild type polyextremophilic B. halodurans in submerged fermentation and its application in pre-bleaching of paper pulps. [Copyright &y& Elsevier]

Abstract: The phytases of thermophilic molds are histidine acid phosphatases that catalyze the hydrolysis of phytic acid (myo-inositol hexakisdihydrogen phosphoric acid) to inorganic phosphate and myo-inositol through a series of myo-inositol phosphate intermediates. Phytases can be produced by thermophilic molds in solid-state and submerged fermentations. The biochemical properties of the phytases from thermophilic molds suggest that they are phosphate ester hydrolyzing enzymes, ranging in molecular mass from 54 to 456kDa depending on the source and are usually active within pH and temperature ranges of 4.5–7.0 and 50–70°C, respectively. Phytases not only degrade phytic acid to increase the availability of phosphorus but also improve the nutritional value of foods by making minerals such as calcium, iron, magnesium and zinc and proteins available for monogastrics. Thermophilic fungal phytases are known to hydrolyze insoluble phytates, to improve the bread-making process, to dephytinize food ingredients and to help in the promotion of plant growth. This review addresses the production, characteristics and multifarious applications of phytases from thermophilic molds. [Copyright &y& Elsevier]

Aims: Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread. Methods and Results: The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air-lift fermenters. Among surfactants tested, Tweens (Tween-20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X-100 inhibited the enzyme production. The mould produced phytase optimally at aw 0·95, and it declined sharply below this aw value. The enzyme production was comparable in air-lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca-alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread. Conclusions: The phytase production by S. thermophile was enhanced in the presence of Tween-80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca-alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high-soluble phosphate. Significance and Impact of the Study: The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air-lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid. [ABSTRACT FROM AUTHOR]

Abstract: Phytase production by a thermophilic mould Sporotrichum thermophile Apinis was investigated in solid state fermentation (SSF) using sesame oil cake as the substrate. Scanning electron microscopy of the fermented sesame oil cake revealed a dense growth of the mould with abundant conidia. Glucose, ammonium sulphate and incubation period were identified as the most significant factors by Plackett–Burman design. The optimum values of the critical components determined by central composite design of response surface methodology for the maximum phytase production were glucose 3%, ammonium sulphate 0.5% and incubation period 120h. An overall 2.6-fold improvement in phytase production was achieved due to optimization. Highest enzyme production (348.76U/g DMR) was attained at a substrate bed depth of 1.5cm in enamel coated metallic trays. The enzyme liberated inorganic phosphate from wheat flour and soymilk with concomitant dephytinization and liberation of soluble inorganic phosphate. [Copyright &y& Elsevier]

Aims: The present investigation is aimed at assessing the suitability of cane molasses as a cheaper carbon and energy source for glucoamylase production using alginate-immobilized Thermomucor indicae-seudaticae. Methods and Results: The culture variables for glucoamylase production were optimized by ‘one-variable-at-a-time’ strategy and response surface methodology (RSM). A high glucoamylase titre was attained when 40 alginate beads ( c. 5 × 106 immobilized spores) were used to inoculate 50 ml of cane molasses (8%) medium in 250-ml Erlenmeyer flasks. Response surface optimization of fermentation parameters (cane molasses 7%, inoculum level 44 alginate beads per 50 ml of medium and ammonium nitrate 0·25%) resulted in 1·8-fold higher glucoamylase production (27 U ml−1) than that in the unoptimized medium (15 U ml−1). Enzyme production was also sustainable in 22 l of laboratory air-lift bioreactor. Conclusions: Cane molasses served as an excellent carbon and energy source for the economical production of glucoamylase, which was almost comparable with that in sucrose yeast-extract broth. The statistical model developed using RSM allowed determination of optimum levels of the variables for improving glucoamylase production. Significance and Impact of the Study: The cost of glucoamylase produced in cane molasses supplemented with ammonium nitrate was considerably lower (€1.43 per million U) than in synthetic medium containing sucrose and yeast-extract (€35.66 per million U). The reduction in fermentation time in air-lift bioreactor with sustainable glucoamylase titres suggested the feasibility of scale up of the process. [ABSTRACT FROM AUTHOR]

Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett–Burman design, three fermentation variables (pH, sucrose concentration, and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production was achieved in shake flasks (17,054 U g−1 DYB) and laboratory fermenter (18,465 U g−1 DYB), respectively. [ABSTRACT FROM AUTHOR]

The production of polygalacturonase (PGase) by Sporotrichum thermophile Apinis in stirred submerged fermentation (SmF) was high in comparison with that in static conditions. Yeast extract (0.25%) and citrus pectin (2%) at pH 7.0 and 45 °C supported a high enzyme production in flasks agitated at 200 rpm. An overall 1.75-fold enhancement in PGase production was achieved as a result of optimization. The enzyme was optimally active at pH 7.0 and 55 °C, and exhibited t1/2 of 4 h at 65 °C. The Km and Vmax values of the enzyme (for pectin) were 0.416 mg ml-1 and 0.52 μmol mg-1 min-1, respectively. The PGase activity was stimulated by Mn2+ and Fe2+, but inhibited strongly by Mg2+, and slightly by Tween 80 and Triton X-100. Among the additives tested, β-mercaptoethanol exerted a strong inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. An increase in the yield of banana, grape and apple juices was recorded due to the treatment of fruit pulps with the mixture of enzymes (pectinase, xylanases and cellulase) of S. thermophile as compared to that with only pectinase. The yield of fruit juices did not increase with enhanced titre of cellulase in the enzyme mixture. [Copyright &y& Elsevier]