Development of an economically competitive Trichoderma-based platform for enzyme production: Bioprocess optimization, pilot plant scale-up, techno-economic analysis and life cycle assessment.

Despite decades of research and industrial applications of Trichoderma reesei, the development of industrially relevant strains for enzyme production including a low-cost and scalable bioprocess remains elusive. Herein, bioprocess optimization, pilot plant scale-up, techno-economic analysis and life-cycle assessment for enzyme production by an engineered T. reesei strain are reported. The developed bioprocess increased in âˆ¼ 2-fold protein productivity (0.39 g.L-1.h-1) and 1.6-fold FPase activity (196 FPU.L-1.h-1), reducing the fermentation in 4 days. Cultivation in a 65-L pilot plant bioreactor resulted in 54 g.L-1 protein in 7 days, highlighting the robustness and scalability of this bioprocess. Techno-economic analysis indicates an enzyme cost of âˆ¼ 3.2 USD.kg-1, which is below to the target proposed (4.24 USD.kg-1) in the NREL/TP-5100-47764 report, while life-cycle assessment shows a carbon footprint reduction of approximately 50% compared to a typical commercial enzyme. This study provides the fundamental knowledge for the design of economically competitive Trichoderma technologies for industrial use.

Application of a recombinant GH10 endoxylanase from Thermoascus aurantiacus for xylooligosaccharide production from sugarcane bagasse and probiotic bacterial growth.

Xylooligosaccharides (XOs) are a promising class of prebiotics capable of selectively stimulating the growth of the beneficial intestinal microbiota against intestinal pathogens. They can be obtained from xylan present in residual lignocellulosic material from agriculture. Thus, in this study we produced XOs by extracting xylan from sugarcane bagasse and hydrolyzing it using the GH10 xylanase from Thermoascus aurantiacus expressed by Pichia pastoris. An alkaline method to extract xylan is described, which resulted in 83.40% of xylan recovery and low amounts of cellulose and lignin. The enzymatic hydrolysate exhibited a mixture of XOs containing mainly xylobiose, xylotriose and xylotetraose. These oligosaccharides stimulated the growth of Lactobacillus casei, L. rhamnosus, L. fermentum and L. bulgaricus strains, which were able to produce organic acids, especially acetic acid. These findings demonstrate the possibility to redirect crop by-products to produce XOs and their use as a supplement to stimulate the growth of probiotic strains.

Identification of a cold-adapted and metal-stimulated ß-1,4-glucanase with potential use in the extraction of bioactive compounds from plants.

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-ß-1,4-mannanase.

ß-Mannanases from the glycoside hydrolase 26 (GH26) family are retaining hydrolases that are active on complex heteromannans and whose genes are abundant in rumen metagenomes and metatranscriptomes. These enzymes can exhibit distinct modes of substrate recognition and are often fused to carbohydrate-binding modules (CBMs), resulting in a molecular puzzle of mechanisms governing substrate preference and mode of action that has not yet been pieced together. In this study, we recovered a novel GH26 enzyme with a CBM35 module linked to its N terminus (CrMan26) from a cattle rumen metatranscriptome. CrMan26 exhibited a preference for galactomannan as substrate and the crystal structure of the full-length protein at 1.85 Å resolution revealed a unique orientation of the ancillary domain relative to the catalytic interface, strategically positioning a surface aromatic cluster of the ancillary domain as an extension of the substrate-binding cleft, contributing to galactomannan preference. Moreover, systematic investigation of nonconserved residues in the catalytic interface unveiled that residues Tyr195 (-3 subsite) and Trp234 (-5 subsite) from distal negative subsites have a key role in galactomannan preference. These results indicate a novel and complex mechanism for substrate recognition involving spatially remote motifs, distal negative subsites from the catalytic domain, and a surface-associated aromatic cluster from the ancillary domain. These findings expand our molecular understanding of the mechanisms of substrate binding and recognition in the GH26 family and shed light on how some CBMs and their respective orientation can contribute to substrate preference.

GH53 Endo-Beta-1,4-Galactanase from a Newly Isolated Bacillus licheniformis CBMAI 1609 as an Enzymatic Cocktail Supplement for Biomass Saccharification.

Galactanases (endo-ß-1,4-galactanases-EC 3.2.1.89) catalyze the hydrolysis of ß-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-ß-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5-8 and 40 °C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75 % activity even after 24 h of incubation at pH 4-10. The enzyme was thermostable, retaining nearly 100 % activity after 1-h incubation at pH 7.0 at 25-45 °C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2 s(-1) mg(-1) mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE(®)1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25 %.

Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo-arabinanase from Bacillus licheniformis.

OBJECTIVES: The use of endo-arabinanase from Bacillus licheniformis (ABNase) for sugarcane saccharification has been evaluated by enzyme immobilization and commercial cocktail supplement with the immobilized heterologous protein. RESULTS: Biochemical characterization of the purified ABNase showed that the catalytic activity was strongly inhibited by 5 mM Cu(2+), Zn(2+) or Fe(3+). The optimum pH and temperature for activity were 5.5-6.5 and 35-40 °C, respectively. The enzyme stability increased 128-fold when immobilized with glyoxyl agarose, and the hydrolysis of pretreated sugar cane biomass increased by 15 % when a commercial enzyme cocktail was supplemented with immobilized ABNase. CONCLUSION: Pectin hydrolysis by recombinant ABNase plays a role in the effective application of enzymatic cocktails for biomass saccharification.