Alternan is a novel functional oligosaccharides which has good prebiotic properties. The content of alternate sugars is less in nature and mainly prepared by microbial fermentation and enzyme conversion. Currently, low activity of alternansucrase prevents its application in largescale. To enhance the enzyme expression, the alternansucrase gene was synthesized and heterologously expressed in Pichia pastoris GS115 for the first time. After 72 h high-density fermentation in 3 L fermenter, the enzyme activities in supernatant reached 34.5 U/ml, which was about 12 times than that of shake flask fermentation, and reached the highest level of recombinant alternansucrase at present. The optimum pH and temperature of recombinant ASR were 6.0 and 55℃, Km and kcat were 31.97 mmol/L and 0.36 s-1, respectively. In addition, alternan was prepared by using sucrose or sucrose and other receptors as substrates at 40°C and pH 5.5. It was identified by TLC that both oligoalternan and polyalternan were produced in the catalytic products. The alteransucrase gene was successfully expressed in P. pastoris for the first time, which will provide a basis for the heterologous expression of alteransucrase and the large-scale preparation of alternan.
DAI Yue
,
WU Dan
,
ZHENG Pu
,
CHEN Pengcheng
. Expression, enzymatic properties and catalytic reaction of alternansucrase in Pichia pastoris[J]. Food and Fermentation Industries, 2022
, 48(17)
: 29
-34
.
DOI: 10.13995/j.cnki.11-1802/ts.030589
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