• Bulletin of the Korean Chemical Society
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• 제14권3호
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• pp.398-403
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• 1993

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1991년도 춘계학술발표대회 논문집
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• pp.225-236
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• 1991

The genes encoding the thermostable $\alpha$-amylase and maltogenic amylase from Bacillus lichenciformis were cloned and expressed in E. coli. The recombinant plasmid pTA322 was found to contain a 3.1kb EcoRI genomic DNA fragment of the thermostable $\alpha$-amylase. The cloned $\alpha$-amylase was compared with the B. licheniformis native $\alpha$-amylase. Both $\alpha$-amylase have the same optimal temperature of $70^{\circ}C$ and are stable in the pH range of 6 and 9. The complete nucleotide sequences of the thermostable $\alpha$-amylase gene were determined. It was composed of one open reading rame of 1,536 bp. Start and stop codons are ATG and TAG. From the amino acid sequence deduced from the nucleotide sequence, the cloned thermostable $\alpha$-amylase is composed of 483 amino acid residues and its molecular weight is 55,200 daltons. The content of guanine and cytosine is $47.46mol\%$ and that of third base codon was $53_41mol\%$. The recombinant plasmid, pIJ322 encoding the maltogenic amylase contains a 3.5kb EcoRI-BamHI genomic DNA fragment. The optimal reaction temperature and pH of the maltogenci amylase were $50^{\circ}C$ and 7, respectively. The maltogenic amylase was capable of hydrolysing pullulan, starch and cyclodextrin to produce maltose from starch and panose from pullulan. The maltogenic amylase also showed the transferring activity. The maltogenic amylase gene is composed of one open reading frame of 1,734bp. Start and stop codons are ATG and ATG. At 2bp upstream from start codon, the nucleotide sequence AAAGGGGGAA seems to be the ribosome-binding site(RBS, Shine-Dalgarno sequence). A putative promoter(-35 and-10 regions) was found to be GTTAACA and TGATAAT. From deduced amino acid sequence from the nucleotide srquence, this enzyme was comosed of 578 amino acid residues and its molecular weight was 77,233 daltons. The content of guanine and cytosine was $48.1mol\%$. The new recombinant plasmid, pTMA322 constructed by inserting the thermostable $\alpha$-amylase gene in the EcoRI site of pIJ322 to produce both the thermostable $\alpha$-amylase and the maltogenic amylase were expressed in the E. coli. The two enzymes expressed from E. coli containing pTMA322 was reacted with the $15\%$ starch slurry at $40^{\circ}C$ for 24hours. The distribution of the branched oligosaccharides produced by the single-step process was of the ratio 50 : 50 between small oligosaccharide up DP3 and large oligosaccharide above DP3.

• 한국식품과학회지
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• 제24권4호
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• pp.371-375
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• 1992

Bacillus licheniformis가 분비하는 ${\alpha}-amylase$를 정제한 후 기질과 금속이온이 효소의 열안정성에 미치는 영향에 대하여 연구하였다. $Ca^{++}$ 이온 및 $B^{+++}$ 이온의 존재시에는 D-value가 7,880 sec 및 2,210 sec로 대조구에 비하여 높은 값을 보였으며 $Ca^{++}$ 이온과 $B^{+++}$ 이온이 동시에 존재할 때는 $D_{85}=24,000\;sec$로 상승효과를 보였다. $Ca^{++}$ 이온 존재시 열불활성화에 대한 활성화에너지 $({\Delta}H{\neq})$는 320.2 kJ/mol이고 $B^{+++}$ 이온의 경우에는 212.9 kJ/mol이었으며 대조구에서는 183.9 kJ/mol 이었다. 기질의 존재하에서는 ${\alpha}-amylase$는 높은 열안정성을 보였는데 30% 전분의 존재하에 $96^{\circ}C$, 30분 열처리한 결과 약 51%의 잔존역가를 검출하였으며 기질에 $Ca^{++}$ 이온을 첨가하였을 때에는 열안정성이 더욱 증가하였다.

• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1696-1701
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• 2010

The ${\alpha}$-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis ${\alpha}$-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.

• Journal of Microbiology and Biotechnology
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• 제22권5호
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• pp.592-599
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• 2012

Bacillus licheniformis ${\alpha}$-amylase (BLA), a thermophilic counterpart of Bacillus amyloliquefaciens ${\alpha}$-amylase (BAA), is an appropriate model for the design of stabilizing mutations in BAA. BLA has 10 more histidines than BAA. Considering this prominent difference, in the present study, three out of these positions (I34, Q67, and P407; located in the thermostability determinant 1 region and Ca-III binding site of BAA) were replaced with histidine in BAA, using the site-directed mutagenesis technique. The results showed that the thermostability of P407H and Q67H mutants had increased, but no significant changes were observed in their kinetic parameters compared to that of the wild type. I34H replacement resulted in complete loss of enzyme activity. Moreover, fluorescence and circular dichroism data indicated a more rigid structure for the P407H variant compared with that of the wild-type BAA. However, the flexibility of Q67H and I34H mutants increased in comparison with that of wild-type enzyme.

• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.898-904
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• 2014

The stability of Bacillus licheniformis alpha-amylase (BLA) under acid condition was enhanced through direct evolution using the error-prone polymerase chain reaction. One beneficial mutation site, H281I, was obtained in BLA. The specific activity of H281I was 161/352 U/mg, which was 62.6/27.5% higher than that of the wild-type (WT) (99/276 U/mg) at pH 4.5/6.5 and $95^{\circ}C$. The pH optimum for H281I was decreased about 1 unit, whereas no significant changes of optimum temperature and thermostability were observed compared with the wild type (WT). The $k_{cat}/K_m$ value of H281I was 1.7-/1.4-fold higher at pH 4.5/6.5, respectively, than that of WT. The structure model analysis indicated that the H281I mutation altered the predicted interaction between the amino acid residues at 281 and 273, thus creating a conducive local environment for substrate binding, as reflected by its decreased $K_m$, and consequently increased the specific activity.

• 한국식품과학회지
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• 제32권2호
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• pp.410-416
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• 2000

고초균과 효모를 혼용 첨가한 고추장을 6개월간 숙성시키면서 pH, 미생물 및 효소활성도의 변화를 조사한 결과는 다음과 같이 요약할 수 있다. pH 변화는 담금 직후에는 $5.29{\sim}5.35$에서 숙성 60일 경에는 $4.71{\sim}4.84$로 낮아졌으며 효모 첨가구에서는 숙성 초기에 pH 강하가 심한 것으로 나타났으나 유의적인 차이는 없었다. 호기성 세균 수는 고초균 첨가시 담금 초기에는 $3.9{\sim}4.2{\times}10^6$ 생균 수였던 것이 숙성 30일 경까지 급속히 증가하였고 그후 서서히 감소되어 숙성 90일 이후에는 일정 수준을 유지하였다. 곰팡이 수는 숙성 30일까지 현저히 증가한 후 감소되어 숙성 60일 이후에는 완만한 수준을 보였고, 고추장 숙성 기간 중 효모 무첨가구에서는 고추장 숙성 30일경을 전후하여 효모를 발견할 수 있었으며 숙성 60일까지 급격히 증가한 후 감소하였다. 효모 첨가구에서는 30일 이후부터 효모 수가 증가하였으며 숙성 60일 이후부터는 감소하였다. ${\alpha}-amylase$와 ${\beta}-amylase$ 활성도는 곰팡이와 고초균을 혼합한 시험구가 비교적 높은 활성을 보여 주었고 효모첨가구에서는 활성이 감소되었다. Acidic protease 활성도는 숙성 30일 이후 급격히 증가하는 경향이 나타났으며 특히 곰팡이와 고초균 혼용구에서 보다 높은 활성도를 보여주었다. 따라서 곰팡이 단용구에 비해 고초균을 혼용 첨가한 고추장이 숙성 중 미생물 수도 많았고 효소 활성도가 높은 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제23권1호
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• pp.7-14
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• 2013

In the Bacillus amyloliquefaciens ${\alpha}$-amylase (BAA), the loop (residues 176-185; region I) that is the part of the calcium-binding site (CaI, II) has two more amino acid residues than the ${\alpha}$-amylase from Bacillus licheniformis (BLA). Arg176 in this region makes an ionic interaction with Glu126 from region II (residues 118-130), but this interaction is lost in BLA owing to substitution of R176Q and E126V. The goal of the present work was to quantitatively estimate the effect of ionic interaction on the overall stability of the enzyme. To clarify the functional and structural significance of the corresponding salt bridge, Glu126 was deleted (${\Delta}$E126) and converted to Val (E126V), Asp (E126D), and Lys (E126K) by site-directed mutagenesis. Kinetic constants, thermodynamic parameters, and structural changes were examined for the wild-type and mutated forms using UV-visible, atomic absoption, and fluorescence emission spectroscopy. Wild-type exhibited higher $k_{cat}$ and $K_m$ but lower catalytic efficiency than the mutant enzymes. A decreased thermostability and an increased flexibility were also found in all of the mutant enzymes when compared with the wild-type. Additionally, the calcium content of the wild-type was more than ${\Delta}E126$. Thus, it may be suggested that ionic interaction could decrease the mobility of the discussed region, prevent the diffusion of cations, and improve the thermostability of the whole enzyme. Based on these observations, the contribution of loop destabilization may be compensated by the formation of a salt bridge that has been used as an evolutionary mechanism or structural adaptation by the mesophilic enzyme.

• 한국식품과학회지
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• 제41권4호
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• pp.369-373
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• 2009

전분액화에 호화찰옥수수전분가 쌍축형 압출성형의 원료로 직접 사용되었다. 그 액화 효과와 체류시간분포를 분석하였다. 내열성 ${\alpha}$-amylase(Bacillus licheniformis로부터 분리)를 함께 첨가하여 사용하였다. 전분액화는 환원당측정, gel permeation chromatography(GPC), 주사전자현미경을 통하여 분석하였다. 배럴온도와 수분함량이 높을수록 환원당 함량은 증가하였고, 호화전분의 경우 생전분보다 더 많은 환원당이 생성되었다. GPC에서 호화전분의 사용에 의한 저분자화 효과는 뚜렷하지 않았으나, 효소 첨가없이 압출성형하는 경우에는 일부 효과가 있는 것으로 나타났다. 주사전자현미경의 미세구조에서는 호화전분을 쓸 경우 그 표면이 더 불규칙하고 침식되는 것을 관찰할 수 있었다. 종합적으로 호화전분의 사용이 전분액화에 효과가 있음을 알 수 있었다. 체류시간분포에서는 호화전분을 원료로 할 경우 그 분산도가 커지는 것으로 나타났다. 이는 호화전분은 압출성형의 흐름이 원만하지 않은 것을 의미하였으나, 배럴온도와 수분함량을 증가시킴에 따라 그 분산도를 감소시킬 수 있음을 알 수 있었다.

• BMB Reports
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• 제40권3호
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• pp.315-324
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• 2007

The novel $\alpha$-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of $Ca^{2+}$ and EDTA. Therefore, KRA acts as a $Ca^{2+}$-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of $Ca^{2+}$ and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its Tm. The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis $\alpha$-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the $\alpha$-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.