| Alpha-amylase is called starch-1,4-dextrinase,and is also named ?-1,4-D-glucanglucoside hydrolase.When acting on starch,it can randomly cut ?-1,4-glucosidic bonds from the inside of starch,polysaccharide or oligosaccharide molecules to produce glucose,maltose and oligosaccharides.Alpha-amylase is widely used in food,medicine,textile,paper and other industrial fields.Ultra-high temperature ?-amylase refers to ?-amylase whose optimum temperature is above 90 ?,which has more superior enzymatic properties,making it more suitable for the spray liquefaction process of starch,improving the efficiency of starch processing production,and reducing production cost.Bacillus subtilis is a type of Gram-positive rod-shaped good-culturing bacteria and a model organism in Bacillus.It has been approved as a food safety microorganism(GRAS)by the US Food and Drug Administration(FDA)and the Ministry of Agriculture of China.Therefore,using the B.subtilis system to produce enzyme preparations has great industrial value.In the early stage of the laboratory,there has been some research and modified on B.subtilis expression system,and it has its own good B.subtilis expression host.In this study,the ultra-high temperature ?-amylase(PFA)gene derived from the extreme thermophilic archaea Pyrococcus furiosus was expressed in B.subtilis,and the enzymatic properties of the recombinant ultra high temperature ?-amylase were determined.Through signal peptide screening,chaperone protein co-expression and heat treatment,it can effectively improve the extracellular enzyme activity of PFA recombinantly expressed in Bacillus subtilis.Finally,on the basis of shake flasks,the fermentation medium components and fermentation conditions were optimized,and the optimized fermentation broth was heat-treated.The main findings are as follows:(1)The ultrahigh temperature ?-amylase gene derived from the extreme thermophilic archaea P furiosus and the expression vector p HY300 PLK were constructed into a recombinant plasmid p HY300PLK-pfa and transformed into the host B.subtilis WS9 for recombinant expression.The extracellular enzyme activity was 18.33 U·m L-1.The enzymatic properties of recombinant ultra-high temperature ?-amylase were explored.The results of enzymological properties show that the optimal reaction temperature of recombinant ultra-high temperature ?-amylase is 100?,the optimal reaction p H is 5.0,and the treatment at 100? for 4 h also retains 60% of the enzyme activity.The recombinant enzyme has Very good thermal stability.(2)In order to improve the recombinant heterologous expression of PFA,173 signal peptides of Bacillus subtilis were screened by applying a high-throughput screening system,and two excellent signal peptides were obtained,including Yfh K of the Sec pathway and another new signal.Peptide Asp B'.They were replaced on the expression vector p HY300PLK-pfa for recombinant construction,and transferred to the host B.subtilis WS9 for fermentation and culture.The results showed that the extracellular enzyme activity of the recombinant strain using Yfh K as the signal peptide was 60.73 U·m L-1,and the extracellular enzyme activity was increased by approximately 3.31 times.The extracellular enzyme activity can reach 119.01 U·m L-1,which is about 6.49 times higher.(3)For the problem that heterothermous expression of thermophilic archaea-derived ?-amylase in mesophilic hosts will generate inclusion bodies,consider using a chaperone co-expression strategy to improve it.Chaperone proteins have functions such as assisting protein folding and keeping intracellular precursor proteins in a transportable state.First,the chaperone protein prs A derived from B subtilis was selected,and the co-expression recombinant plasmid p HY300PLK-prs A-Asp B'-pfa was constructed and transferred into the host B.subtilis WS9,which was fermented in shake flask for 60 h in TB medium.Live up to 134.15 U·m L-1.Compared with the initial expression,it is about 7.32 times higher.Then select the chaperone proteins Pfefoldin? and PPlase derived from the thermophilic archaea P furiosus.The CRISPR/Cas9 gene editing technology was used to insert the genes of Pfefoldin? and PPlase into the B.subtilis WS9 genome,respectively,and the host bacteria B.subtilis WS9din? and B.subtilis WS9 PPlase were obtained.The recombinant plasmid p HY300PLK-prs AAsp B'-pfa was transformed into these two host bacteria,and a recombinant strain containing both chaperone protein combinations prs A/Pfefoldin? and prs A/PPlase was obtained.After shaking flask fermentation under the same conditions,the extracellular enzyme activities reached 145.54 U·m L-1 and 156.67 U·m L-1,respectively,which were about 7.94 times and 8.55 times higher than the initial expression.(3)During the fermentation culture of recombinant bacteria,it was found that with the increase of shake flask fermentation culture time(?300 h),the enzyme activity gradually increased.The ultra-high temperature enzyme has good thermal stability.In order to accelerate the effective soluble expression of the ultra-high temperature ?-amylase,heat treatment of the fermentation broth containing fermentation broth can significantly improve the ultra-high temperature ?-amylase in B subtilis Soluble expression.The recombinant strain WS9PPlase(p HY300PLK-prs A-Asp B'-pfa)was fermented and cultured for 72 h,and the extracellular enzyme activity was measured to reach 233.26 U·m L-1,and the heat-treated(90°C water bath heated for 15min)bacteriacontaining fermentation broth sample was centrifuged to obtain the supernatant.At this time,the ?-amylase activity in the supernatant was 1456.65 U·m L-1.Centrifuge the sample for 120 h,thoroughly remove the supernatant,add 1 m L of deionized water to suspend the bacteria,heat treat at 90°C for 15 minutes,and centrifuge to obtain the aqueous suspension.The enzyme activity of each part was measured 673.19 U·m L-1,the enzyme activity in the water suspension was 1507.42 U·m L-1,and the total enzyme activity reached 2108.61 U·m L-1.(4)The enzymatic properties of the recombinant ultra-high temperature ?-amylase were explored,and the fermentation conditions of the recombinant bacteria were optimized at the shake flask level.The results of enzymatic properties showed that the optimal reaction temperature for recombinant ultra-high temperature ?-amylase was 100°C,the optimal reaction p H was 5.0,and it was treated at 100°C for 4 h,and 60% of the enzyme activity was retained.The recombinant enzyme has very good thermal stability.The shake flask optimization results showed that the optimal medium combination composition was: a composite nitrogen source consisting of 18 g·L-1 Xiwang Soy Peptone and 9 g·L-1 Angel yeast extract,5 g·L-1 glucose as carbon source,10 m M Concentrations of metal ions of Ca2+ and 3 m M Al3+,p H of initial culture medium at p H 7.5,fermentation temperature of 33°C.The fermentation conditions of recombinant bacteria B.subtilis WS9PPlase(p HY300PLK-prs A-Asp B'-pfa)were optimized at the shake flask level.The results of the shake flask optimization showed that the optimal medium composition was: a compound nitrogen source composed of 18 g·L-1 Xiwang soy peptone and 9 g·L-1 Angel yeast extract powder,and 5 g·L-1 glucose Carbon source,metal ion concentration of 10 m M Ca2+and 3 m M Al3+,initial culture medium p H of p H 7.0,fermentation culture temperature of 33°C,After 60 h of fermentation,the extracellular enzyme activity was 282.60 U·m L-1,which is higher than before optimization It was about 1.80 times.Heat treatment of the fermentation broth containing cells,the extracellular enzyme activity can reach 1302.79 U·m L-1.During the same period of shake flask fermentation,the extracellular enzyme activity was increased by about 71.08 times compared with the initial expression.Fermentation culture in 3-L tank for 120 h,the extracellular soluble enzyme activity can reach 3806.69 U·m L-1. |
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