Heterologous Expression Of Novel ?-mannanase Gene From Thermophilic Bacteria And Its Preliminary Study On The Protein Feeding Value

?-mannanase is an important hemicellulase that catalyzes the hydrolysis of mannan to oligosaccharides or monosaccharides.Mannan is an important component of plant hemicellulose.It is rich in plant cell walls such as soybean meal and rapeseed meal.And it's the main anti-nutritional factor in feed.It can hydrolyze mannan in the feed material to oligosaccharides or monosaccharides when adding mannanase.It can significantly reduce the high chyme viscosity of the polysaccharides such as mannan in the digestive tract of animals.Therefore,it can promote the digestion and absorption of nutrients in the intestinal tract of animals,prevent animal diseases and enhance animal health.Moreover,mannanase is a green feed additive because it is safe and non-toxic and does not cause adverse reactions with animals.In addition,mannanase is widely used in many industries such as food,paper,and medicine,and has a huge market demand.Therefore,it is particularly important and necessary to excavate the novel mannanase gene with excellent performance and explore its production method and application value.The gene?Tnman?sequence suspected of?-mannanase?KUK22456.1?in the thermophilic bacteria Thermotoga naphthophila genome based on sequence homology search of the mannomosidase gene?Tpman?derived from the thermophilic bacteria Thermotoga petrophila,The Tnman gene sequence is 1959 bp in length and encodes the protein TnMAN.The theoretical molecular weight of TnMAN is 72 kDa,which contains 650 amino acids.The amino acid sequence similarity between TnMAN and TpMAN protein is as high as 95%,and the Tnman gene expression product TnMAN has not been reported.Optimize the Tnman gene codon and then fully synthesize the optimized Tnman,then,the expression vector PET21a-tn was constructed,and the recombinant plasmid was transformed into E.coli BL21?DE3?to obtain a Tnman gene heterologous expression recombinant strain.The heterologous expression of the gene Tnman was successfully induced by the recombinant strain with IPTG at a final concentration of 1 mM.It was proved that the TpMAN has mannanase activity and is a novel mannanase by analyzing the function of the target protein TnMAN.The recombinant strain expressing the Tnman gene heterologously was subjected to shake flask fermentation and upper tank fermentation test,and the results showed that the enzyme production amount was 123 U/mL and2356 U/mL.It was showed that the fermentation in the upper tank was about twice the yield of the shake flask fermentation enzyme by comparing the enzyme production efficiency per unit strain.Therefore,the recombinant strain heterologously expressing the Tnman gene can stably and efficiently utilise the enzyme under high-density culture conditions.These conclusions provide a basis for research on the future application of this strain to the production of this novel mannannase.Furthermore,the substrate specificity and enzymatic properties of the novel mannanase TnMAN protein expressed by the Tnman gene were also investigated.The results showed that the highest substrate specificity of the new mannanase was locust bean gum?LBG?,followed by konjac flour and guar gum.The results of enzymatic analysis showed that the optimum reaction temperature of the new mannanase?TpMAN?was 80?,there is no loss of enzyme activity at 70?for 12 h,and 50%retention at 90?for 3 h..Its optimum pH is 7.0,and it has good stability at pH5.0^pH 9.0.The metal ions Co²⁺?Ca²⁺?EDTA,etc.with a final concentration of 1mM have obvious activation effects,and Fe²⁺?Fe³⁺?Mn²⁺have obvious inhibitory effects.When Locust bean gum?LBG?was used as the substrate,the kinetic parameters Km and Vmax were 6.28 mg/mL and 0.063 mg/min respectively.It shows that the novel?-mannanase has excellent performance.It has good substrate specificity and high catalytic hydrolysis activity for the main anti-nutritional factor locust bean gum in feed ingredients.Moreover,the new?-mannan enzyme type has strong thermal stability,which is favorable for its application in production.This study analyzed the tolerance of the enzyme to trypsin degradation to explore the application value of the novel mannanase in feed.In addition,the in vitro degradation test of the enzyme feedstock palm kernel meal was also explored.The results showed that TpMAN retained 85%of enzyme activity in 2 mg/mL trypsin solution after incubation at 55?for 16 h.This indicates that the novel mannanase TpMAN has good trypsin tolerance and can be stably deposited in animals,so it is beneficial to better degrade the anti-nutritional factor mannan in animal digestive tract feed.The test of the animal diet containing 40%palm kernel showed that the in vitro digestibility of palm kernel meal was the highest when adding 10 U/g mannanase,which was 39.4%higher than that of the control group.And the protein digestibility of palm kernel meal increased with the increase of the amount of mannanase added.This indicates that the novel mannanase?TpMAN?can not only effectively degrade the anti-nutritional factor mannan in the feed material,but also promote the digestion and absorption of protein in the feed material.Therefore,the addition of the novel mannanase preparation to animal feed has important significance for promoting animal growth,enhancing animal immunity and preventing animal diseases.