No abstract

Glucose oxidase (GOD, EC.1.1.3.4) specifically catalyzes the reaction of β-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability of the current commercial GOD is a key limiting factor preventing its widespread application. In the present study, amino acids closely related to the thermostability of glucose oxidase from Penicillium notatum were predicted with a computer-aided molecular simulation analysis, and mutant libraries were established following a saturation mutagenesis strategy. Two mutants with significantly improved thermostabilities, S100A and D408W, were subsequently obtained. Their protein denaturing temperatures were enhanced by about 4.4 °C and 1.2 °C, respectively, compared with the wild-type enzyme. Treated at 55 °C for 3 h, the residual activities of the mutants were greater than 72%, while that of the wild-type enzyme was only 20%. The half-lives of S100A and D408W were 5.13- and 4.41-fold greater, respectively, than that of the wild-type enzyme at the same temperature. This work provides novel and efficient approaches for enhancing the thermostability of GOD by reducing the protein free unfolding energy or increasing the interaction of amino acids with the coenzyme.

Corn flour was prepared by wet-milling with the treatment of neutral protease and the gelatinization, thermal and rheological properties were analyzed. Tortilla was prepared with enzyme treated corn flour (ECF) and additives (xanthan gum and cassava starch) and the properties were analyzed. Compared with dry-milling corn flour (DCF) and wet-milling corn flour (WCF), the ECF had less average particle size (16.74 μm), higher peak viscosity and higher final viscosity of 2997 cP and 3300 cP, respectively. The thermal properties showed that ECF had higher ∆H and lower To, Tp and Tc. The G′ of ECF gel (6%, w/w) was higher than that of DCF gel and WCF gel. Dynamic viscoelastic measurement indicated that the tortillas made of ECF had lower G′ and G″ over the frequency range (0.1–100 rad/s) after adding xanthan gum and cassava starch. The gel structure of tortillas made of ECF was homogeneous in distribution of pores. The gelatinization, thermal and rheological properties of corn flour were improved by addition of neutral protease. The addition of xanthan gum and cassava starch helped to make the tortilla with porous structure and good sensory quality.

Resistant starch (RS) type 2-high-amylose corn starch (HACS) was subjected to simultaneous hydrothermal (25% moisture content, 90 °C for 12 h) and microwave (35% moisture content, 40 W/g microwaving for 4 min) treatment and zein (at a zein to treated starch ratio of 1:5, 50 °C for 1 h) to improve its resistance to enzymolysis. Scanning electron microscopy (SEM) highlighted the aggregation and adhesion of the composite. The average particle size of the composite (27.65 μm) was exceeded that of both the HACS (12.52 μm) and the hydrothermal and microwave treated HACS (hydro-micro-HACS) (12.68 μm). The X-ray diffraction results revealed that the hydro-micro-HACS and composite remained B-type, while their crystallinity significantly decreased to 16.98% and 12.11%, respectively. The viscosity of the hydro-micro-HACS and composite at 50 °C was 25.41% and 35.36% lower than that of HACS. The differential scanning calorimetry (DSC) results demonstrated that the composite displayed a new endothermic peak at 95.79 °C, while the weight loss rate and decomposition temperature were 7.61% and 2.39% lower than HACS, respectively. The RS content in HACS, the hydro-micro-HACS, and composite was 47.12%, 57.28%, and 62.74%, respectively. In conclusion, hydrothermal and microwave treatment combined with zein provide an efficient physical strategy to enhance the RS type 2-HACS.