Screening, optimization of process parameters and scale-up of native and recombinant thermophilic Xylanases and their application in chicken feed hydrolysis.

Abstract

Lignocellulosic biomass is a renewable raw material that has gained industrial interest due to its abundance, low cost, and potential to mitigate greenhouse gas emissions. Biomass is treated with various microbial enzymes to produce desired products under ideal conditions. Thermophilic microorganisms are excellent sources of thermostable enzymes that can tolerate extreme conditions. Optimized xylanases can be produced through genetic engineering, and recombinant DNA techniques. The biotechnological potential of xylanases from thermophilic microorganisms is discussed and the ways they are being optimized and expressed for industrial applications. Monogastric animal farming relies on grain feedstocks with non-starch polysaccharides (NSP’s) and anti-nutritive factors that cause adverse effects like increased digesta viscosity and nutrient inaccessibility which leads to reduced feed conversion, energy metabolism, and growth. Exogenous enzymes have been used to reduce viscosity and increase nutrient absorption in poultry and pigs. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their ability to modulate the composition of the gut microbiota. This study aimed to isolate and screen potential xylanolytic fungi from soil and tree bark samples in South Africa and to determine their growth conditions for maximum xylanase production. The highest xylanase activity was produced by Trichoderma harzianum. The enzyme with a molecular weight of 72 kDa retained >70% activity after 4 h at pH 6.0 and 70°C. The study also identified multiple isoforms of xylanase, which could be beneficial for animal feed and biofuel industries. The xylanase was purified from the submerged culture and displayed maximum xylanolytic activity at pH 6.0 and 65°C and the enzyme was activated by Fe2+, Mg2+, and Zn2+. Enzyme production was then optimized for maximal xylanase production strain using the Plackett-Burman Design (PBD) and Box Behnken Design (BBD), screening, and optimization design strategies, respectively. Xylanase production was enhanced to 153.80 U/ml by BBD representing a 3.99-fold increase and a 2.24- fold increase, respectively compared to the preliminary one-factor-at-a-time (OFAT) activity of 68.7 U/ml. The experimental design effectively provided conditions for the production of an acidic enzyme based on pH and incubation time. This is an exciting prospect for the application of enzymes in animal feed improvement (pH 5.0). The Geobacillus stearothermophilus glycoside hydrolase family 10 xylanase, endoxylanase XT6, is a promising candidate for industrial application. This gene was cloned and expressed and the xylanase was applied as an additive to locally produced chicken feeds. Optimization of cell lysis and expression conditions led to enhanced recombinant XT6 xylanase production. The recombinant XT6 xylanase was purified using cobalt chromatography, resulting in a 43 kDa protein with 15.69-fold purity. Cellulose, hemicelluloses, and lignin are the primary sources of fermentable sugars in lignocellulosic feedstock. Carbohydrate-active enzymes which can help release functional compounds from the carbohydrate matrix, such as phenolics are used to modify polysaccharides for industrial purposes. However, it should be noted that the primary action of carbohydrate enzymes like cellulases or xylanases is specific to the carbohydrate structure and may involve the hydrolysis of polysaccharides like xylan or other complex carbohydrate-lignin compounds,rather than being directly responsible for the release of phenolic compounds. Corn, with its antioxidant potential, is used in animal feed production, thus improving its quality for animal feed supplementation is crucial. The crude and purified T. harzianum xylanases as well as the recombinant XT6 xylanase were applied to locally produced chicken feeds in an experimental BBD design to optimize hydrolysis to monosaccharides and XOS. The Response Surface Methodology (RSM) results showed that higher (8.05 U/ml) levels of reducing sugars were produced for the crude T. harzianum xylanase and starter feed, than on the grower feed (3.11 U/ml). Treatment with the purified T. harzianum and recombinant XT6 xylanases produced lower levels of reducing sugars with similar levels for both feed types of starter feed (2.81 U/ml) and (2.98 U/ml), compared to the grower feed (2.41 U/ml) and (2.62 U/ml), respectively. Profiling of the hydrolysis products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed that the chicken feed enzymatic hydrolysates contained a range of monosaccharides (mannose, glucose and galactose) and XOS, with xylobiose being the predominant XOS. Toxicity studies showed that the higher dilutions of the feed enzymatichydrolysates were not toxic to HEK293 cells. Therefore, the T. harzianum and recombinant XT6 xylanase are appropriate for application in the feed industry to produce XOS. These results are promising for future studies and application in the poultry feed industry as additives. The novelty of this study was the identification and characterization of a thermostable xylanase from a South African T. harzianum isolate, the application of experimental design to optimize its production and as well as that of a recombinant XT6 xylanase. The recombinant XT6 xylanase exhibited high yields in bioreactor production with activity superior to that of a commercial xylanase preparation, further emphasizing its potential for commercialization through scalingup techniques and its industrial application. Moreover, the enzymes investigated in this researchhold promise for the production of prebiotics in animal feed applications.