低聚半乳糖(galactooligosaccharide,GOS)是一种具有天然属性的功能性低聚糖,因其优良的益生功能以及在高温和低pH下较高的稳定性而被广泛应用于婴幼儿奶粉等食品中,其主要由β-半乳糖苷酶以乳糖为底物进行生产。为了将具有更高转化率的β-半乳糖苷酶应用于食品领域,该研究首次将Bacillus circulans来源的β-半乳糖苷酶β-Gal-Ⅱ在Bacillus subtilis中进行了异源表达,并对其酶学性质、GOS的制备进行了测定,同时为了降低其工业化应用成本,对重组菌在摇瓶及3-L罐的发酵条件进行了优化。结果表明,重组β-Gal-Ⅱ制备GOS的最适作用条件为50 ℃、pH 5.0,该条件下GOS转化率在8 h可达最高值60.32%;在摇瓶的发酵条件优化中,以安琪酵母粉和玉米浆干粉各7.5 g/L为复合氮源时,总酶活力最高,可达6.82 U/mL,是TB发酵的1.5倍,单一氮源发酵的2~2.5倍;在3-L罐上进行了33、37 ℃两个温度的发酵优化,37 ℃发酵产量优于33 ℃,最终总酶活力可达138.3 U/mL,是摇瓶发酵的20.3倍。该研究为GOS的工业化生产提供技术基础。

Galactooligosaccharide (GOS) is a kind of functional oligosaccharide with natural properties. It is widely applied in infant formula and other foods due to its excellent probiotic function and high stability at high temperature and acidic pH. It is mainly produced by β-galactosidase using lactose as substrate. In order to enhance the application in food field of β-galactosidase from Bacillus circulans, namely β-Gal-Ⅱ, which exhibited higher conversion rate than most commercial enzymes, this study heterologously expressed β-Gal-Ⅱ in Bacillus subtilis for the first time. Its enzymatic properties and performance in GOS preparation were characterized. Moreover, the fermentation conditions of β-Gal-Ⅱ in shake flasks and 3 L bioreactor were optimized to reduce the cost of its industrial application.
In this study, the coding gene of β-Gal-Ⅱ was codon optimized according to the preference of B. subtilis and chemically synthesized. Then the synthesized DNA fragment was ligated into plasmid pHY300PLK, a constitutive vector without signal peptide for B. subtilis expression system. SDS-PAGE results showed that both fermentation supernatant and the cell rupture supernatant had obvious bands of target protein. This result might be caused by the non-classical protein secretion in B. subtilis. The optimum temperature and pH of β-Gal-Ⅱ were tested to be 60 ℃ and 5.0 using the fermentation crude enzyme solution. It exhibited good pH stability in the range of pH 5.0-7.0, and it maintained 95% activity after incubated at 40 ℃ for 24 h. The half-life of β-Gal-Ⅱ at 50 ℃ was about 3 h, but lost almost all activity after placed at 60 ℃ for 30 min. Using 400 g/L lactose as substrate, the yield of GOS synthesized by recombinant β-Gal-Ⅱ at 50 ℃ was slightly higher than that at 40 ℃ and 60 ℃. The highest yield reached 60.32% after 8 h of reaction at 50 ℃, which was superior to most of reported β-galactosidases.Although β-Gal-Ⅱ exhibited excellent GOS conversion rate and short reaction cycle, its expression level in TB shake flask fermentation was only 4.55 U/mL, which resulted in extremely high costs of adding enzyme to meet the required dosage of enzyme during enzymatic preparation of GOS. To reduce the cost of enzyme preparation, fermentation condition of β-Gal-Ⅱ recombinant strain was optimized. Firstly, the type, concentration composition and ratio of nitrogen source were optimized at the shake flask level. Eleven kinds of nitrogen sources were selected to replace the yeast powder and tryptone in TB. SDS-PAGE gel electrophoresis analysis and enzyme activity determination were performed to screen the best fermentation nitrogen source. The results showed that, when 7.5 g/L of yeast extract fermentation (YEF, Angel) and 7.5 g/L corn steep powder (Angel) were used as the compound nitrogen source, the target protein band was thickest and the total enzyme activity reached the highest value of 6.82 U/mL, which was 1.5 times that of TB fermentation and 2-2.5 times that of any single nitrogen source fermentation.
After optimizing the fermentation nitrogen source at the shake flask level, the fermentation in 3 L bioreactor was continued to be enlarged and cultured. Considering that the temperature has a certain influence on the growth of recombinant bacteria and enzyme production, temperatures of 33 ℃ and 37 ℃ were adopted to optimize fermentation temperature in the 3 L bioreactor. The optimum fermentation condition temperature was also judged by SDS-PAGE and enzyme activity determination. The OD₆₀₀ values of the bacteria under the two fermentation temperatures were basically the same, and both reached the highest value of about 120 at about 84 h of fermentation, indicating that the fermentation temperature did not have much effect on the concentration of the bacteria. However, the enzyme activity under fermentation temperature of 37 ℃ was much higher than that under 33 ℃. SDS-PAGE also showed that the target protein band under 37 ℃ fermentation was thicker than that under 33 ℃. Finally, the highest enzyme activity reached 138.29 U/mL in 3 L bioreactor with a high concentration of 8.87 mg/mL of target protein, which was 20.3 times that of the enzyme activity of 6.82 U/mL in shake flask fermentation. The scale-up from shake flasks to 3 L bioreactor was successfully realized, providing a technical basis for the industrial production of GOS.