The β-mannanase gene (1,029 nucleotide) from Bacillus subtilis MAFIC-S11, encoding a polypeptide of 342 amino acids, was cloned and expressed in Pichia pastoris. To increase its expression, the β-mannanase gene was optimized for codon usage (mannS) and fused downstream to a sequence-encoding modified α-factor signal peptide. The expression level was improved by 2-fold. This recombinant enzyme (mannS) showed its highest activity of 24,600 U/mL after 144-h fermentation. The optimal temperature and pH of mannS were 50 °C and 6.0, respectively, and its specific activity was 3,706 U/mg. The kinetic parameters V max and K m were determined as 20,000 U/mg and 8 mg/mL, respectively, representing the highest ever expression level of β-mannanase reported in P. pastoris. In addition, the enzyme exhibited much higher binding activity to chitin, chitosan, Avicel, and mannan. The superior catalytic properties of mannS suggested great potential as an effective additive in animal feed industry.

In this study, the activity of an α-galactosidase obtained from Penicillium janczewskii zalesk was improved via modifying its gene by error-prone PCR and DNA shuffling. The mutated DNA was ligated to pBGP1, an autonomous-replicating vector, which was subsequently transformed into Pichia pastoris X-33. The expressed enzyme activities were measured after single colonies were cultured in yeast-peptone-dextrose medium in deep-well plates. After two rounds of screening, two mutants with higher activity were obtained. By PCR analysis, four mutation sites (S167G, P455L, N637S, and P490L/P490H) were found in these two variants (mutant-59 and mutant-8). Mutant-59 showed the highest activity at pH 5.0 and 40 °C with an increased V(max) value of 769 μmol/min and the specific activity of 667 U/mg against p-nitrophenyl α-D-galactopyranoside. The two mutant enzymes also showed similar resistance to the metal ions of Cu(2+), Fe(2+), and Zn(2+). In a 10-L fermenter, the supernatant enzyme activity reached the maximum of 550.2 U/mL upon the methanol induction for 96 h. This fermentation activity of the mutant was improved approximately two more folds than the wild type α-galactosidase. This mutant of α-galactosidase is prospective in feed manufacturing as feed additives to improve nutrient digestibility in monogastric animals.

In order to improve some characteristics of a β-1,3-1,4-glucanase from Bacillus subtilis MA139, directed evolution was conducted in this study. After error-prone PCR, the β-1,3-1,4-glucanase gene, glu-opt, was cloned into the vector pBGP1 and transformed into Pichia pastoris X-33 to construct a mutant library. Three variants named as 7-32, 7-87, and 7-115 were screened from 8000 colonies. Amino-acid sequence analysis showed that these mutants had one or two amino-acid substitutions (7-32: T113S, 7-87: M44V/N53H, and 7-115: N157D). The variants were over-expressed in P. pastoris by methanol induction. After purification of the enzyme proteins, the characteristics of the variants were analyzed in detail. It indicated that these mutant enzymes had broader ranges of pH value and better pH stability than the wild-type enzyme. The mutant enzyme 7-87 had the best ability to tolerate an acid environment (pH 2.0), while the wild-type enzyme had no activity under this condition. Moreover, all these mutants demonstrated improved thermal stability. In particular, the mutant enzyme 7-32 had residual enzymatic activity of 60% and 40% after being incubated at 80 °C and 90 °C for 10 min. While, the wild-type enzyme had no residual enzymatic activity after being incubated at 80 °C for 4 min. In addition, the mutant enzymes had better tolerance to some chemicals than the wild-type enzyme. The improved stability could enhance the prospects for this enzyme to have use in the feed industry to reduce the effects of the anti-nutritional factor β-glucan.