• Journal of Microbiology and Biotechnology
• /
• 제25권1호
• /
• pp.33-43
• /
• 2015

GAM-1 and GAM-2, two themostable glucoamylases from Aspergillus niger B-30, possess different molecular masses, glycosylation, and thermal stability. In the present study, the effects of additives on the thermal inactivation of GAM-1 and GAM-2 were investigated. The half-lives of GAM-1 and GAM-2 at 70℃ were 45 and 216 min, respectively. Data obtained from fluorescence spectroscopy, circular dichroism spectroscopy, UV absorption spectroscopy, and dynamic light scattering demonstrated that during the thermal inactivation progress, combined with the loss of the helical structure and a majority of the tertiary structure, tryptophan residues were partially exposed and further led to glucoamylases aggregating. The thermal stability of GAM-1 and GAM-2 was largely improved in the presence of sorbitol and trehalose. Results from spectroscopy and Native-PAGE confirmed that sorbitol and trehalose maintained the native state of glucoamylases and prevented their thermal aggregation. The loss of hydrophobic bonding and helical structure was responsible for the decrease of glucoamylase activity. Additionally, sorbitol and trehalose significantly increased the substrate affinity and catalytic efficiency of the two glucoamylases. Our results display an insight into the thermal inactivation of glucoamylases and provide an important base for industrial applications of the thermally stable glucoamylases.

• 한국식품과학회지
• /
• 제16권4호
• /
• pp.398-402
• /
• 1984

Rhizopus oryzae 가 생산(生産)하는 glucoamylase 의 각종기질(各種基質)에 대(對)한 분해반응(分解反應)을 검토(檢討)하였다. Glucoamylase I 과 II 는 amylose, amylopectin, glycogen, 가용성 전분, pullulan, maltose, maltotriose, maltotetriose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose를 가수분해(加水分解)하였으나, ${\alpha}-cyclodextrin$, ${\beta}-cyclodextrin$, sucrose, raffinose, 젖당은 가수분해(加水分解)하지 못하였다. $37^{\circ}C$, 32시간(時間)의 반응(反應)에서 glucoamylase I 은 amylopectin, 가용성 전분, amylose를 거의 100% 분해하였고 glycogen 만을 88%정도 분해 하였으나, glucoamylase II 는 공시기질(供試基質) 4 종(種)을 거의 100% 분해하였다. Glucoamylase I 과 II 의 반응생성물질(反應生成物質)은 glucose 만이었고 ${\alpha}-glucosyltransferase$ activity는 없었다. Glucoamylase I 과 II 는 생찹쌀 전분을 가장 잘 분해(分解)시키나 생감자 전분, 생미숙 바나나 전분, 생칡 전분, 생참마 전분, raw high amylose corn starch의 분해능(分解能)은 glucoamyase I 에 비(比)하여 생전분(生澱粉)의 분해력(分解力)이 강(强)하였다.

• Journal of Microbiology and Biotechnology
• /
• 제20권2호
• /
• pp.383-390
• /
• 2010

A newly isolated active producer of raw-starch-digesting amyloytic enzymes, Rhizopus microsporus var. chinensis CICIM-CU F0088, was screened and identified by morphological characteristics and molecular phylogenetic analyses. This fungus was isolated from the soil of Chinese glue pudding mill, and produced high levels of amylolytic activity under solid-state fermentation with supplementation of starch and wheat bran. Results of thin-layer chromatography showed there are two kinds of amyloytic enzymes formed by this strain, including one $\alpha$-amylase and two glucoamylases. It was found in the electron microscope experiments that the two glucoamylases can digest raw corn starch and have an optimal temperature of $70^{\circ}C$. These results signified that amyloytic enzymes secreted by strain Rhizopus microsporus var. chinensis CICIM-CU F0088 were types of thermostable amyloytic enzymes and able to digest raw corn starch.

• 한국버섯학회지
• /
• 제9권2호
• /
• pp.53-58
• /
• 2011

In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3'- and 5'-RACE PCR and RT-PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

• BMB Reports
• /
• 제28권5호
• /
• pp.375-381
• /
• 1995

Two forms of glucoamylase (GI and GII) from starch-grown Lipomyces kononenkoae CBS 5608 mutant were purified to apparent homogeneity by means of ultrafiltration, Sephacryl S-200 gel filtration and DEAE Sephadex A-50 chromatography. The apparent molecular weight was calculated as ca. 150 kDa for GI and ca. 128 kDa for GII, respectively. Both enzymes were glycoproteins with isoelectric points of 5.6 (GI) and 5.4 (GII). They had a pH optimun of 4.5 and were stable from pH 5 to 8. The temperature optimum for both enzymes was $60^{\circ}C$, but they were rapidly inactivated above $70^{\circ}C$. The $K_m$ values toward starch were estimated to be 6.57 mg per ml for GI and 4.52 mg per ml for GII, and the $V_{max}$ values were 16.28 ${\mu}M$ per mg for GI and 32.25 ${\mu}M$ per mg for GII, respectively. The $K_m$ and $V_{max}$ values of GII for ${\alpha}-$ or ${\beta}-cyclodextrin$ were estimated to be 0.15 mg per ml and 2.0 mg per ml, respectively ($K_m$) and 1.02 ${\mu}M$ per mg or 1.02 ${\mu}M$ per mg, respectively ($V_{max}$). Neither enzyme exhibited pullulanase activity but they released only glucose from starch or cyclodextrin. Amino acid analysis indicated that both glucoamylases were enriched in proline and acid amino acids. Glucoamylase GII strongly cross-reacted with a monoclonal antibody raised against GI enzymes, and the two enzymes shared very similar amino acid composition. Western blot analysis indicated that L. kononenkoae CBS 5608 mutant produced two forms of glucoamylase on starch, and that synthesis of them was subject to glucose repression.

• Applied Biological Chemistry
• /
• 제28권4호
• /
• pp.233-238
• /
• 1985

Glucoamylase를 $ZrO_2$로 피복된 96% porous glass에 azo-linkage를 형성시켜 결합하게 한후, 2.5% glutaraldehyde로 처리하여 효소를 고정화시켰다. 효소기질로는 용해도가 높고 점도가 낮은 30% enzyme thinned cornstarch (dextrose equivalent 값 : 24)를 사용하여 plug flow-column reactor에서 연속반응시켰다. 반응 최적 pH는 수용성효소의 5.0보다 alkaline 쪽으로 기울어져 7.0으로 나타났고, 고정화반응에 따라 열안정성이 높아지고 $40{\sim}60^{\circ}C$에서 최적 온도범위를 가리키며, Km값은 수용성 효소의 1.25mM보다 낮은 1.04mM값을 보여 주었다. 따라서, pH 7.0, $45^{\circ}C$에서 160시간 동안 corn starch를 기질로 효소반응을 시켜 glucose 90.3%, maltose 8.0%인 DE값 94.0인 전분당분해산물을 획득할 수 있었다.

• 한국미생물·생명공학회지
• /
• 제17권5호
• /
• pp.529-532
• /
• 1989

A strain of Streptomyces sp. (YS-221-B) extracellularly produced an inhibitory substance for $\alpha$-D-Glucosidase. The substance was purified 96-fold from culture filtrate by dialysis, heat treatment, adsorption on active carbon, Bio-Gel P-10 and Sephadex G-75 column chromatography with yield of 9.2%. The substance was stable in pH range from 7.0 to 11.0 at 37$^{\circ}C$, and a treatment at 10$0^{\circ}C$ for 20 min diminished only 15% of the original activity. The inhibitor was not inactivated by the treatment of $\alpha$-, $\beta$-amylases, glucoamylases, trypsin and chymotrypsin but inactivated by pyoteases from Streptomyces griseus and Tritirachium album.

• 한국미생물·생명공학회지
• /
• 제16권6호
• /
• pp.489-493
• /
• 1988

Starch로부터 ethanol을 직접적으로 발효 생산할 수 있는 새로운 효모 균주를 개발하고자 glucoamylase 생성균으로 알려진 Saceharomyces diastaticus의 glucoamylase gene을 cloning vector를 사용하지 않고 S, cerevisiae에 transformation시켰다. Li$_2$SO$_4$, 처리로써 competent화 한 S, cerevisiae의 Intact cells을 recipient로 하여 BamHI으로 partial digestion한 S, diastaticus의 chromosomal DNA를 transformation시키고 starch를 유일한 탄소원으로 함유한 최소 배지상에서 starch 자화능을 marker로 하여 transformant를 선별한 결과, 8.5$\times$$10^{-7}$ 빈도로 transformant를 얻었다. Transformant의 특성을 recipient 및 donor와 비교하기 위해 copper resistance와 당 발효능을 조사한 결과, donor인 S, diastaticus와 동일한 성질로서 표현된 maltose와 starch 발효능을 제외하고는 800ppm 농도까지 생육 가능한 copper resistance와 galactose 발효능 등에 있어서는 recipent와 동일하게 나타났다. 또한 transformant가 생성하는 glucoamylase의 그 작용에 있어서의 최적온도와 최적pH를 조사하여 본 바 각각 pH5.0, 50C로서 donor의 glucoamylase와 동일함을 알 수 있었다.

• Food Science and Biotechnology
• /
• 제14권6호
• /
• pp.860-865
• /
• 2005

A gene for a putative glucoamylase, stg, of a hyperthermophilic archae on Sulfolobus tokodaii was cloned and expressed in Escherichia coli. The recombinant glucoamylase (STGA) had an optimal temperature of $80^{\circ}C$ and was extremely thermostable with a D-value of 17 hr. The pH optimum of the enzyme was 4.5. Being different from fungal glucoamylases, STGA hydrolyzed maltotriose (G3) most efficiently. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis showed that the enzyme existed as a dimer. STGA was stable enough to hydrolyze liquefied com starch to glucose in 4 hr at $90^{\circ}C$ with a yield of95%. Comparison of the $k_{cat}$ values for the hydrolysis and the reverse reaction at $75^{\circ}C$ and $90^{\circ}C$ indicated that glucose production by STGA was more efficient at $90^{\circ}C$ than $75^{\circ}C$. Therefore, STGA showed great potential for application to the industrial glucose production process due to its high thermostability.

• 미생물학회지
• /
• 제18권4호
• /
• pp.161-171
• /
• 1980

A mutant strain having increased productivity of both enzymes, protease and amylase, was obtained from A. flavus KU 153, isolatd from South Korea for its high protease production by successive ultra-violet light irradiation, Two glucoamylases from the mutant strain selected were purified from wheat branculture by successive salting out, followed by dialysis and column chromatography, and their characteristics were compared with those of the wild strain. Glucoamylase production of the mutant selected was increased about 3.3 times compared with the wild strain, and 2.1 times compared with the parental strain, ${\alpha}-amylase$ activity of the mutant selected was about 2 times hugher than that of the wild strain or the parental strain. Protease and cellulase productivities of the muant selected were all alike compared with those of the highly proteolytic mutant, the parental strain. Therefore, it was considered that the back mutation on the protease production did not occurred in the formation process of the glucoamylase producing mutant. Total activities of glucoamylase I and II from the mutant selected were 2.86 and 3.65 times higher compared with those from the wild strain, respectively. Considering the optimal pH-thermal stability and Km-Vmax value of glucoamylase I and II from both strains, wild and mutant, it was deduced that the characteristics of glucoamylase I and II from the wild strain did not altered during the mutation process. Therefore, it was concluded that the selected mutant did not induce the formation of another glucoamylase isozyme, or the changes in the characteristics of the glucoamylase, but induce the productivity of the same glucoamylase I and II by the action of regulatory gene.