Cloning and Characterization of a Galactomannan-degrading Enzyme Gene in Pichia pastoris

[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone that could hydrolyze guar gum was obtained through the construction and functional screening of a soil genome library. Sequence analysis indicated that the 1485-bp clone encodes a 494-amino acid protein with a relative molecular mass of 53 949 kD, containing a cellulose-binding domain. The recombinant plasmid pHBM731 was generated by inserting the optimized target gene into a Pichia pastoris expression vector pHBMg05 that was transformed into three Pichia pastoris strains, GS115, KM71 and SMD1168. The biochemical properties of the enzyme were assessed. [ Result] The cloned galactonumnan （GM）-degrading enzyme was expressed and secreted by Pichia pastoris GSll5. High cell density fermentation was induced in recombi- nant Pichia pastoris at 25 and 28 ~C ; a higher enzyme activity was observed at an induction temperature of 28 ~C. The optimal temperature for the recombinant en- zyme is 60 ~C, and the optimal pH is 6.6. The enzyme activity was 38.61 U under optimal conditions. Over 50% of the enzyme activity was maintained under the optimal conditions after 9 h. Under the optimal conditions, the effect of metal ions on enzyme activity was analyzed. Ca2 ＋ , Fe2 ＋ and Li ~ slightly enhanced enzyme activity, while Mn2＋ and Co2＋ had little effect. Enzyme activity was modestly suppressed by Mg2~ , K~ and Na＋ , but considerably suppressed by Ag2~ and Zn2~ , with Cu2 ＋ showing the strongest inhibitory effects. [ Conclusion] A novel GM-degrading enzyme expressed by soil yeast was cloned, which can potentially be used in industrial applications to obtain eommereially useful guar gum-degradation products.

Preliminary Study on Selection and Identification of a Mannanase-producing Strain and Its Enzymatic Properties

[Objective] This study aimed to identify a maunanase-producing strain isolated from soil. [Method] With kanjac powder as the substrate, a man- nanase-producing dominant strain was iselated from the soil samples collected from Kunyu Mountain by using plate selection method. Sequence analysis of the 16SrDNA fragment of the strain was conducted, and the strain was identified as Bacillus subtilis. Fermentation conditions and enzymatic characteristics were studied preliminarily. [ Result] Experimental result showed that enzyme yield of this strain was different in different medium and in the same medium at different tempera- ture. Enzyme yield of this strain in LB medium was higher when incubated at 27 ℃ than at 30 ℃ ; however, incubation at 30 ℃ was more conducive to the enzyme production than incubation at 27 ℃ in SOC medium. The optimal reaction pH was 7.0 and the optimal reaction temperature was 55 ℃ for enzyme production of this strain. When the temperature was above 55 ℃, enzyme activity declined sharply with the raise of temperature. Under the optimum conditions, enzyme activity could achieve 95.3 U. [ Conclusion] This study provided reference for the industrial application of degradation products of mannan.