Locust bean gum (LBG) was employed to screen mannanase-producing bacteria. The bacterium with highest mannanase ability was identified as Paenibacillus cookii. It revealed highest activity (6.67 U/mL) when cultivated in 0.1% LBG with 1.5% soytone and 0.5% tryptone after 4 days incubation at 27 °C. Its mannanase was purified to electrophoretical homogeneity after DEAE-Sepharose and Sephacryl S-100 separation. The purified mannanase, with an N-terminus of GLFGINAY, had pH and temperature optimum at 5.0 and 50 °C, respectively, and was stable at pH 5.0−7.0, ≤50 °C. It was strongly activated by β-mercaptoethanol, dithiothreitol, cysteine, and glutathione, but inhibited by Hg 2+ , Cu 2+ , Zn 2+ , Fe 3+ , PMSF, iodoacetic acid, and EDTA. According to substrate specificity study, the purified mannanase had high specificity to LBG and konjac.

To obtain a Pichia pastoris mutant with an Escherichia coli phytase gene, which was synthesized according to P. pastoris codon preference, a mature phytase cDNA of E. coli being altered according to the codons usage preference of P. pastoris was artificially synthesized and cloned into an expression vector of pGAPZαC. The final extracellular phytase activity was 112.5 U/mL after 72 h of cultivation. The phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The yield, purification fold, and specific activity were 63.97%, 26.17, and 1.57 kU/mg, respectively. It had an optimal pH and temperature of 4.0-6.0 and 50 °C, respectively, and was stable at pH 3.0-8.0 and 25-40 °C. The purified recombinant phytase was resistant to trypsin, highly inhibited by Cu(2+), Zn(2+), Hg(2+), Fe(2+), Fe(3+), phenylmethylsulfonyl fluoride, and N-tosyl-l-lysine chloromethyl ketone, but activated by Mg(2+), Ca(2+), Sr(2+), Ba(2+), glutathione, ethylenediaminetetraacetic acid, and N-ethylmaleimide. It revealed higher affinity to calcium phytate than to other phosphate conjugates.

Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Grampositive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 μg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.