The Study Of Hyperthermophiles Acid-Stable α-Amylase From Hyperthermophilic Archaeon Thermococcus Siculi HJ21

a-Amylase(EC 3.2.1.1), endo-acting enzyme that hydrolyzes starch by cleaving-1,4-glucosidic linkages at random sites, is one of the most important commercial enzymes widely used in starch-processing, brewing, alcohol production, textile, and other industries. The most thermostable a-amylase in industry is produced from Bacillus licheniformis. This enzyme operates optimally at 90℃and pH 6, and it requires addition of calcium ion (Ca2+) for its thermostability. Hyperthermophiles that optimally grow at temperatures above 80℃, have attracted many researchers’ attention as they are a source of enzymes with thermoactive and outstanding thermostability. In this paper, we report the biological characteristics and molecular identification of a hyperthermophilic archaeon (strain HJ21), the production and characterization of an thermoactive a-amylase produced by this strain, the cloning and sequence analysis of a-amylase gene, the expression in E. coli and the characterization of recombinant a-amylase, and cultivation optimization of high cell density and high expression for the recombinant E. coli. Main study results as following:1. The identification and characterization of extremely anaerobic hyperthermophilic archaeon strains HJ21, isolated from a deep-sea hydrothermal vent, has been studied. The cells were round to slightly irregular cocci,1 to 1.2μm in diameter. The isolate grew between 60 and 94℃with an optimum around 88℃. The pH range for growth was 5.0 to 9.0, with an optimum around 6.5～7.0. The NaCl range for growth was 10 to 50 g·L-1, with an optimum around 20 g·L-1. Strain HJ21 grew much better in the medium with yeast extract, peptone, tryptone, casein and HJ21 was capable of growing on starch, maltose, glucose, sucrose, cellobiose, lactose, glycogen. The growth of strain HJ21 could be enhanced by elemental sulfur. Cells were also grown in the absence of elemental sulfur; however, the growth was very slow. The growth of strain HJ21 could be enhanced by elemental sulfur. The amount of elemental sulfur added in medium in shaken cultures was less than the medium in stable cultures. On the basis of 16S rDNA sequence comparisons, in combination with morphology, physiological characteristics, it is identified as Thermococcus siculi. At present the research about thermoactive a-amylase from Thermococcus siculi have not reported.2. The production and characterization of a-amylase from T. siculi HJ21 has been studied. Maximum enzyme production was achieved after 9 h cultivation. The temperature of a-amylase production were between 60 and 90℃with an optimum around 80℃. The pH range for production of amylase was 5.0 to 9.0, with an optimum around 7.5. The NaCl range for production of amylase was 10 to 50 g·L-1, with an optimum around 2.5 g·L-1. Inoculation amount of the strain was 5%(v·v-1). Addition of yeast extract, peptone, glycogen, starch and maltose enhanced enzyme production. The strain HJ21 produced extracellular thermostable and acid-stable a-amylase. The molecular weight of the enzyme was estimated to be 51.4 KDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The a-amylase exhibited maximal activity at 95℃. The half-life of the enzyme was 5 h at 90℃, more than 40 of the enzyme activity remained after 2h of incubation at 100℃. The enzyme did not require Ca2+ for thermostability. The optimal pH of a-amylase was observed at 5.0, and more than 80% amylase activity of the maximum enzyme activity was detected at pH 4.5. The enzyme was shown to be stable in the broad range of pH 5.0 to 7.0 (at 80℃2h). The enzyme was activated by Mg2+, Sr2+, K+, Na+. It was inhibited by Cu2+、Pb2+、Hg2+、Zn2+、Al3+. N-Bromosuccinimide (at 1 mmol·L-1 and 5 mmol·L-1) showed a significant repression to enzyme activity.3. Thisα-amylase gene was cloned and its nucleotide sequence analysed The gene sequence between the conserved region was acquired by PCR using the primers designed based on the sequence of that of hyperthermophilic archaeon deposited in the GenBank. The upstream and downstream of T. siculi HJ21α-amylase gene was acquired by siteFind-PCR. After Sequence analysis, the complete T. siculi HJ21α-amylase gene about 1374bp was obtained through the juncture of them. The similarity between theα-amylase of T. siculi HJ21 and that of T. hydrothermalis and T. sp. OGL-20P was over 95%. Four highly conserved regions, possibly forming the active site, have been identified in a-amylases. These regions were also found in T. siculi HJ21 a-amylase gene and showed great similarities with those of other Thermococcales a-amylases secreted by thermophilic organisms. The gene sequence of was submitted to http://swissmodel. expasy. org to predict the structure of the a-amylases. T. siculi HJ21 a-amylase was single and classicα/βstructure. There were problems of degenerate and preference of codon usage in a-amylase through analysis the sequence of amimo acid.4. The gene encoding a-amylase from T. sicili HJ21 was expressed in E. coli and the characterization of the recombinant enzyme was studied. The expression plasmid pEt-28a-His6-amy was constructed. The plasmid was transformed into E. coli BL-21(DE3). The recombinant a-amylase was purified to homogeneity by heat treat, DEAE-sepherose ion exchange chromatography, Ni affinity chromatography and gel recovery. The molecular weight of the enzyme was estimated to be 50 KD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum temperature for the activity was 90℃. More than 90% and 70%α-amylase activity of the maximum enzyme activity was detected at 95℃and 100℃respectively. The enzyme did not require Ca2+ for thermostability.82%、77% and 69% of the enzyme activity remained after 5 h of incubation at 80,90 and 100℃respectively. The amylase exhibited maximal activity at pH 5.0 to 5.5 and was stable in the range of pH 5.0 to 7.5 at 100℃,1 h. The enzyme was activated by K+, Na+. It was inhibited by Hg2+、Pb2+、Al3+、Cu2+、Fe3+、Zn2+. N-Bromosuccinimide (at 1 mmol·L-1 and 5 mmol·L-1) showed a significant repression to enzyme activity. The Km and Vmax vales of the amylase for soluble starch were 45 mg·mL-and 9 mg·ml-1·min-1, respectively. The amylase hydrolyzed soluble starch to produce maltose and maltotriose as the main products.5. Cultivation optimization of high cell density and high expression for the recombinant E. coli in shaking condition. Inoculation amount 1%(v·v-1), liquid filling quantity,20%(v·v-1);pH,7.0;temperature,37℃;inducement opportunity, OD6001.0, amount of IPTG,1 mmol·L-1; inducement time,4 h. The optimal concentration in medium were:glucose,0.5g·L-1; glycerol,0.5g·L-1;peptone,10 g·L-1; yeast extract,5 g·L-1; KH2PO4,10 mmol·L-1;MgSO4,0.6 g·L-1. Response surface design was used to optimize the fermentation condition for a-amylase production by the recombinant E. coli. Facters such as liquid filling quantity, pH, inducement opportunity and inducement time were selected and tested at three levers, respectively. The optimum conditions of the parameters were liquid filling quantity 23.57%, pH 6.4, inducement opportunity 4.03 h and inducement time 4.16 h. After Optimization, specify amylase activity was 10.4 U·mg-1, increases by 1.56 times, and the amount of the protein expression was 8.75%, increases by 1.64 times.