Gene Cloning, Identification And Expression Of Isoamylase From Bacillus Lentus CICIM304

Isoamylase （EC3.2.1.68） is one of the starch debranching enzymes that hydrolyzesα-1,6-glucosidic linkages in glycogen, amylopectin and their phosphorylase limit dextrins toyield amylose and oligosaccharides. Although isoamylase are found to be produced out ofboth botanical and microbial sources, microbial strains are often used for commercial purposedue to their high efficiency, easy extract and low cost. Compared with pullulanase, isoamylasehas three advantages: both endo-and exo-cleavage activity, higher efficient cleavage on α-D-（1,6）-glucoside linkages and less inhibition by its main product maltose. However, it is notavailable for industrial use either because of its low yield or because of its instability underthe conditions of current starch processing.Therefore, in this study, a Bacillus expression system was engineered to inactivateamylase and one of proteases, in order to meet the requirement for isoamylase expression.Then a novel isoamylase gene from Bacillus lentus CICIM304was cloned and expressed as afunctional enzyme. Moreover, a semi-rational designed direct evolution guided by structuralinformation was carried out to improve the catalytic properties of isoamylase.The main resultsare as follows:1. Bacillus licheniformis expression system was improved by genetic engineering.temperature-sensitive plasmid vectors were constructed to inactivate amyL and aprEgene of the host. An engineered Bacillus licheniformis with amyL and aprE gene inactivatedwas constructed. The gene inactivation was based on a highly temperature-sensitive plasmidvector. amyL and aprE, coding α-amylase and one of proteases, respectively, were clonedfrom genomic DNA of B. licheniformis. Two artificial nonfunction-copies of the genes wereconstructed by insertion of antibiotic genes. Then, the constructed delivery plasmidspNZTATN and pNZTPTS, carrying the artifical copies surrounded by DNA fragments thatflank the desired insertion site were transformed into B. licheniformis. After two-roundrecombinations stimulated by temperature shifting, the obtained mutant strain, B.licheniformis APD1had99.1%and65%reduction in extracellular amylase activity andprotease activity, respectively.2. The gene encoding isoamylase in Bacillus lentus CICIM304was cloned,characterized and functionally expressed in E. coli. As isoamylase gene in Bacillus lentusCICIM304has never been reported, a new strategy including MS/MS and inverse-PCRtechniques was adopted. Firstly, the purified enzyme was separated by SDS-PAGE followedby in-gel digestion of trypsin. The peptides released were subjected to nanoLC-ESI-Q-TOF/MS/MS analysis. Then degenerate primer DNAs were designed based on thecharacterized peptides sequences to amplify part of the gene. Genome-walking was performedby using inverse-PCRs and at the end of this experiment, a2658ORF of isoamylase encodinggene was identified, whose product had885amino acids with less than40%identity tomicrobial isoamylase ever reported. Finally, the gene was expressed in E. coli. by using thevector pET-28a （+） and the highest intracellular isoamylase activity was17.6±0.5U/mL. 3. The recombinant isoamylase was purified and characterized. The recombinantisoamylase produced by E. coli was purified by affinity chromatography, which showed asingle band on SDS-PAGE with molecular mass of around100kDa. The biochemicalproperties of the recombinant enzyme were characterized. Its maximal activity occurred at70oC, and1h of incubation at70oC would make it lose merely10%of its maximal activity.The enzyme exhibited good stability between pH6.0and8.5with an optimal activity at pH6.5. Ca²⁺、Mg²⁺、Mn²⁺、Na⁺、K⁺and Co²⁺slightly enhanced the enzyme activity as chloridesalts. Zn²⁺, Fe3+and Sn²⁺reduced the activity evidently to less than50%of control levels.EDTA, citrate and PMSF were not inhibitory, on the contrary,2mol/L urea and30mmol/LSDS led to significant loss of its activity. Maltose and α-cyclodextrin up to a concentration of20%and20mmol/L, respectively, did not influence the activity of the enzyme. A4%inhibition of the activity was observed in the presence of50mM α-Cyclodextrin.4. Semi-rational designed site-derected mutations guided by structural informationwere carried out to improve the catalytic properties of isoamylase. Asp509, His514andGlu609in Region I, III and IV were proved to be essential amino acids by site-directedmutagenesis. Replacement of His698had merely slight effects on activity. The sites near theessential amino acids were chosen as the candidates for mutation for the reason that theseamino acids were located in non-conserved regions so as not to affect the active/catalytic sitesdirectly. On the other hand, mutation of highly conserved region would lead to significantchange in structure which might blow the function of the enzyme strongly to a completeinactivation. Kmvalue of5mutants was reduced, from which R505P and G608V had animprovement in Vmaxof11%and25%, and their specific activity was also raised by13%and33%, respectively.5. Isoamylase was efficiently expressed in engineered B. licheniformis with amyLand aprE gene inactivated. Expression and secretion of the isoamylase in bacillus strainswere under the control of an expression cassette including an artificial bifunctional promoterPQ and S signal peptide region of B. licheniformis amyl gene. In Bacillus subtilis, the highestisoamylase expression level obtained was74U/mL in shaking flask. The expression plasmidpHYQSIA was transformed into B. amyloliquefaciens and B. licheniformis. The functionalexpression of the isoamylase in the two hosts was testified by preparation of maltose fromstarch. Recombinant enzymes obtained88%and92%of conversion yields, respectively,when they was used cooperatively with fungal malto-genic amylase. The results demonstratedthat isoamylase had been functionally expressed in the two hosts as the conversion yieldsobtained by enzymes of parent strain was only around60%. Isoamylase was functionallyexpressed in the two-genes inactivated mutant strain and the higest α-amylase productionlevels obtained was221U/mL under shake flask fermentation. The scale up fermentation wasperformed in a15L fermenter with batch form, and the highest expression level obtained was2140U/mL.