The Study On The Preparation Of Aspergillus Niger Spores As Biocatalysts Of Glucose Oxidase And The Method For Detection Of Glucose Oxidase Activity

Glucose oxidase is widely used in food industry, livestock industry and pharmaceutical industry. Conventional industrial process currently employed for the production of glucose oxidase is based on the use of mycelial inoculum of Aspergillus niger. Fungal fermentation broths using mycelial vegetative forms becomehighly viscous, leading to difficulties in aeration andmixing of the fermentation medium. Moreover, separation and purification of the glucose oxidase is complex, easy to lose enzyme activity and greatly cost. Aspergillus niger spores can be used as a enzyme reservoir of glucose oxidase which is stored at -20℃ for 3 months without losing any activity. So we can develop Aspergillus niger spores as a catalyst in the catalytic reaction of β-D-glucose to gluconic acid, which has numerous advantages like simple culture medium in gredients, easy to separate converted products, simple process and easy to apply in industry. This paper aims to develop Aspergillus niger spores as a catalystin the transformation reaction of β-D-glucose to gluconic acid based on GOD activity in Aspergillus niger spores. In addition, we will do some preliminary study on related issues in applications. The details as follows:(1) To get the preparation method using the Aspergillus niger spores to catalyze glucose to gluconic acid. Glucose oxidase activity in Aspergillus niger spores is best without spores germination. So it’s necessary to inhibit spore germination. Spore wall has a natural protective effect against glucose oxidase catalyzing the substrate. So spores need to be permeabilized. Glucose oxidase activity in Aspergillus niger spores was determined by titration under different conditions. We respectively treated spores with 3% of Citral with freezing-thawing for two days and spores with 3% of Citralwith fresh spores and found the former got higher enzyme activity. The concentration of 109 spores/mL generally got higher enzyme activity than 108spores/ mL under the same shaking culture time and spores treatment conditions.(2) Piezoelectric method to determine glucose oxidase activity. Piezoelectric sensor can sensitively response to the process that glucose oxidase specifically catalyzes the hydrolysis of β-D-glucose to gl uconic acid. So we build the method to detect activity of GOD. First, the kinetic parameter was calculated by changing the substrate concentration. To determine the Michaelis constant of glucose oxidase, we kept enzyme concentrtion constant at 23.75 U and glucose concentration varied from 2 mg/mL to 40 mg/mL. As was determined, we found the initial reaction rate had a linear relationship with glucose oxidase concentration. The Km value was estimated to be 6.01 mg/mL for glucose which was close to the values reported in the literature and a corresponding maximum initial rate (Vmax) was 33.28 Hz/min. Then, we established calibrated equations between enzyme concentration and frequency shift. Glucose oxidase concentration remained unchanged and the piezoelectric sensor read frequency shift within 6 min at different glucose oxidaseconcentration. The initial rateυ0) was defined as slop of response curve of frequency shift within 6 min. The linear relationship between enzyme concentration and initial reaction rate was υ0=1.027 [E]+5.214 (R2=0.995, n= 6). If we knew the initial reaction rate, we would know the glucose oxidase concentration. As was measured by the piezoelectric sensor, the lower enzyme concentration limit was 4.75 U. Using samples spiked to test recovery rate, we calculated recovery rate is between 94.06% to 99.34% and the relative standard deviation is less than 4.3%, which indicated that the activity of glucose oxidase measured by piezoelectric sensing method is accurate and reliable.(3)To determine the content of gluconic acid by piezoelectric sensing method. Piezoelectric sensing method respectively determined frequency shift of spores without any treatment, spores washed five times, spores freezing-thawing for 2 days, spores treated with 1% of Citral, spores treated with 2% of Citral, spores treated with 3% of Citral and spores treated with 3% of Citral with freezing-thawing for 2 days.We found there was a liner relationship between frequency shift and the content of gluconic acid. The piezoelectric sensor responsed maximum frequency shift when it determined spores treated with 3% of Citral with freezing-thawing for 2 days, which meant generating more gluconate. The author respectively measured spores treated with 3% of Citral with freezing-thawing for 2 days by piezoelectric sensing method, titration and enzyme kit method, which found difference value in three methods’s results was less than 50 μg/mL. The titration method had larger standard deviation. The result showed that taking into account the the enzymes kit method much expensive and titration big error in the measurement, piezoelectric sensing method propose todetermine production of gluconic acid for a real-time monitoring and online with advantages of simplicity for operation and less cost.