Pub Date : 2022-07-29DOI: 10.20473/jbiome.v1i1.35859
Wuryanti Handayani, Nasrul Amaliyatun Naja, Muhamad Kiki Afindia Joenata, A. A. I. Ratnadewi
Uricase is an enzyme that degrades uric acid into allantoin. One of the uricase sources is obtained from chicken species (Gallus gallus domesticus) liver which are broiler and native chicken. This study aims to determine the maximum uricase activity in broiler and native chicken liver. The uricase activity was obtained by measuring the uric acid concentration as uricase substrate using spectrophotometric method and wavelength at 291 nm. Uricase isolation was carried out into extraction process, ammonium sulfate fractionation (0-60% saturation of ammonium sulfate), and dialysis. During isolation process, centrifugation speed was also optimized to obtain the maximum uricase crude extract and uricase activity. The molecular weight of uricase was also determined by SDS PAGE. The result showed that the highest uricase activity remained using centrifugation speed of 15,000 rpm. The optimum uricase fraction for broiler chicken liver was obtained at 20-40% saturation of ammonium sulfate with uricase activity was 1.854 x 10-2 U/mg, and the uricase fraction for native chicken liver was obtained at 40-60% saturation of ammonium sulfate with uricase activity was 2.496 x 10-2 U/mg. The optimum fraction for uricase production and isolation is carried out to the dialysis process. The optimum uricase activity of broiler chicken liver crude extract was 4.921 x 10-4 U/mg, the uricase fraction was 3.989 x 10-3 U/mg, and the dialysate was 5.120 x 10-3 U/mg. While the native chicken liver crude extract was 2.980 x 10-4 U/mg, the uricase fraction was1.415 x 10-2 U/mg, and the dialysate was 1.753 x 10-2 U/mg. The molecular weight of the uricase was around 35 kDA according to the SDS PAGE result.
{"title":"Isolation and Characterization of Uricase Produced from Chicken Liver","authors":"Wuryanti Handayani, Nasrul Amaliyatun Naja, Muhamad Kiki Afindia Joenata, A. A. I. Ratnadewi","doi":"10.20473/jbiome.v1i1.35859","DOIUrl":"https://doi.org/10.20473/jbiome.v1i1.35859","url":null,"abstract":"Uricase is an enzyme that degrades uric acid into allantoin. One of the uricase sources is obtained from chicken species (Gallus gallus domesticus) liver which are broiler and native chicken. This study aims to determine the maximum uricase activity in broiler and native chicken liver. The uricase activity was obtained by measuring the uric acid concentration as uricase substrate using spectrophotometric method and wavelength at 291 nm. Uricase isolation was carried out into extraction process, ammonium sulfate fractionation (0-60% saturation of ammonium sulfate), and dialysis. During isolation process, centrifugation speed was also optimized to obtain the maximum uricase crude extract and uricase activity. The molecular weight of uricase was also determined by SDS PAGE. The result showed that the highest uricase activity remained using centrifugation speed of 15,000 rpm. The optimum uricase fraction for broiler chicken liver was obtained at 20-40% saturation of ammonium sulfate with uricase activity was 1.854 x 10-2 U/mg, and the uricase fraction for native chicken liver was obtained at 40-60% saturation of ammonium sulfate with uricase activity was 2.496 x 10-2 U/mg. The optimum fraction for uricase production and isolation is carried out to the dialysis process. The optimum uricase activity of broiler chicken liver crude extract was 4.921 x 10-4 U/mg, the uricase fraction was 3.989 x 10-3 U/mg, and the dialysate was 5.120 x 10-3 U/mg. While the native chicken liver crude extract was 2.980 x 10-4 U/mg, the uricase fraction was1.415 x 10-2 U/mg, and the dialysate was 1.753 x 10-2 U/mg. The molecular weight of the uricase was around 35 kDA according to the SDS PAGE result.","PeriodicalId":371756,"journal":{"name":"Journal of Bio-Molecule Research and Engineering","volume":"144 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2022-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123476223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-07-29DOI: 10.20473/jbiome.v1i1.35856
Evi Umayah Ulfa
Cytochrome P450 monooxygenase (CYP71AV1) is a crucial enzyme in the artemisinin biosynthesis pathway. This enzyme oxidized Amorpha 4,11 diene to produce artemisinic acid. This study aimed to in silico design high-level expression of CYP71AV1 in the E.coli system. In silico techniques are highly suitable for designing protein recombinant production before entering the laboratory. The amino acid sequence of CYP71AV1 was back-translated to the DNA sequence and adapt to E.coli codon usage by using Gene Designer. The DNA sequence of optimized CYP71AV1 was analyzed using Rare Codon Analysis to assess the expression efficiency in E.coli. The protein solubility prediction was determined using the SoDoPe tool. The optimized CYP71AV1 was determined to have a CAI 0.81, a GC content of 53.08 %, CFD with low frequency, and no negative cis or repeat elements. The result of the probability of solubility of CYP71AV1 was 0.6207 when expressed in E.coli. The MBP fusion partner can be used to increase the solubility of CYP71AV1. The in silico results showed the possibility of high-level protein expression of optimized CYP71AV1 in the E.coli system.
{"title":"In Silico Design Gene Encoding CYP71AV1 for Expression in Escherichia coli","authors":"Evi Umayah Ulfa","doi":"10.20473/jbiome.v1i1.35856","DOIUrl":"https://doi.org/10.20473/jbiome.v1i1.35856","url":null,"abstract":"Cytochrome P450 monooxygenase (CYP71AV1) is a crucial enzyme in the artemisinin biosynthesis pathway. This enzyme oxidized Amorpha 4,11 diene to produce artemisinic acid. This study aimed to in silico design high-level expression of CYP71AV1 in the E.coli system. In silico techniques are highly suitable for designing protein recombinant production before entering the laboratory. The amino acid sequence of CYP71AV1 was back-translated to the DNA sequence and adapt to E.coli codon usage by using Gene Designer. The DNA sequence of optimized CYP71AV1 was analyzed using Rare Codon Analysis to assess the expression efficiency in E.coli. The protein solubility prediction was determined using the SoDoPe tool. The optimized CYP71AV1 was determined to have a CAI 0.81, a GC content of 53.08 %, CFD with low frequency, and no negative cis or repeat elements. The result of the probability of solubility of CYP71AV1 was 0.6207 when expressed in E.coli. The MBP fusion partner can be used to increase the solubility of CYP71AV1. The in silico results showed the possibility of high-level protein expression of optimized CYP71AV1 in the E.coli system.","PeriodicalId":371756,"journal":{"name":"Journal of Bio-Molecule Research and Engineering","volume":"16 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2022-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133068485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bioindustries often involve biochemical processes that occur at higher temperatures. However, most proteins, including enzymes, lose their structural integrity and functionality at higher temperatures. Thus, thermostable enzymes from thermophilic microorganisms are best suited candidates for successful bioprocessing under such conditions. Indonesia is one of the best study sites for performing bioprospecting of thermostable enzyme-producing thermophilic microorganisms due to the numerous hot springs. To explore the biodiversity of thermophilic microorganisms with potential industrial applications, we isolated and characterized thermophilic bacteria from the Cangar hot spring, Batu, East Java, Indonesia. One isolate (CGR-1) showed growth at 60°C and was identified as Bacillus subtilis subsp. inaquosorum based on 16s rRNA gene sequencing followed by bioinformatic analysis. This is the first report on the isolation of Bacillus subtilis subsp. inaquosorum CGR-1 from Indonesia, especially from a hot spring environment. This isolate showed cellulolytic and amylolytic activity at 50°C, which would encourage further exploration on the industrial and environmental applications.
{"title":"Isolation and Characterization of Thermophilic Bacillus subtilis subsp. inaquosorum CGR-1 from Cangar Hot Springs","authors":"Almando Geraldi, Aulia Azzahra, Dimas Aryq Ijlal Wafi, Febriani Sukma Maghfirotul Chasanah, Lillah Asritafriha, Rizki Amaliah Zain, Us Watun Nurul Khasanah","doi":"10.20473/jbiome.v1i1.35860","DOIUrl":"https://doi.org/10.20473/jbiome.v1i1.35860","url":null,"abstract":"Bioindustries often involve biochemical processes that occur at higher temperatures. However, most proteins, including enzymes, lose their structural integrity and functionality at higher temperatures. Thus, thermostable enzymes from thermophilic microorganisms are best suited candidates for successful bioprocessing under such conditions. Indonesia is one of the best study sites for performing bioprospecting of thermostable enzyme-producing thermophilic microorganisms due to the numerous hot springs. To explore the biodiversity of thermophilic microorganisms with potential industrial applications, we isolated and characterized thermophilic bacteria from the Cangar hot spring, Batu, East Java, Indonesia. One isolate (CGR-1) showed growth at 60°C and was identified as Bacillus subtilis subsp. inaquosorum based on 16s rRNA gene sequencing followed by bioinformatic analysis. This is the first report on the isolation of Bacillus subtilis subsp. inaquosorum CGR-1 from Indonesia, especially from a hot spring environment. This isolate showed cellulolytic and amylolytic activity at 50°C, which would encourage further exploration on the industrial and environmental applications.","PeriodicalId":371756,"journal":{"name":"Journal of Bio-Molecule Research and Engineering","volume":"2 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2022-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116979668","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-07-29DOI: 10.20473/jbiome.v1i1.35855
Lailatul Fithri, Mamik Damayanti, Anna Kartika Ayu
Corncobs are rich in hemicellulose, which has very important applications in the food industry and biofuels. Hemicellulose is a heteropolysacharide which contains hexosan such as glucan, mannan, galactan and pentosan such as xylan and arabinan. The aims of this research are determining the optimum condition of hemicellulose isolation and identifying enzymatic degradation products of hemicellulose. Hemicellulose is extracted from corn cobs using various NaOH concentrations and extraction times. Acetic acid was added to the mixture after hemicellulose A reflux process, whereas hemicellulose B was precipitated with ethanol 96%. Enzymatic hydrolysis is carried out using xylanolytic enzyme from a recombinant of E. coli DH5α. The yield of hemicellulose is nearly 64.74% (w/w) using NaOH 4 M for 2 h of extraction time. Based on High Performance Liquid Chromatography data indicating that the enzymatic hydrolysis products of hemicellulose A are xylose and arabinose. While xylose, arabinose, and xylooligosaccharide are hemicellulose B and unextracted hemicellulose hydrolysis products.
{"title":"Isolation and Enzymatic Degradation of Hemicellulose from Corncobs Waste","authors":"Lailatul Fithri, Mamik Damayanti, Anna Kartika Ayu","doi":"10.20473/jbiome.v1i1.35855","DOIUrl":"https://doi.org/10.20473/jbiome.v1i1.35855","url":null,"abstract":"Corncobs are rich in hemicellulose, which has very important applications in the food industry and biofuels. Hemicellulose is a heteropolysacharide which contains hexosan such as glucan, mannan, galactan and pentosan such as xylan and arabinan. The aims of this research are determining the optimum condition of hemicellulose isolation and identifying enzymatic degradation products of hemicellulose. Hemicellulose is extracted from corn cobs using various NaOH concentrations and extraction times. Acetic acid was added to the mixture after hemicellulose A reflux process, whereas hemicellulose B was precipitated with ethanol 96%. Enzymatic hydrolysis is carried out using xylanolytic enzyme from a recombinant of E. coli DH5α. The yield of hemicellulose is nearly 64.74% (w/w) using NaOH 4 M for 2 h of extraction time. Based on High Performance Liquid Chromatography data indicating that the enzymatic hydrolysis products of hemicellulose A are xylose and arabinose. While xylose, arabinose, and xylooligosaccharide are hemicellulose B and unextracted hemicellulose hydrolysis products.","PeriodicalId":371756,"journal":{"name":"Journal of Bio-Molecule Research and Engineering","volume":"41 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2022-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128242173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-07-29DOI: 10.20473/jbiome.v1i1.35861
Asni Putri, Galih Ayhusta Laras, Ismi Aulia Syafira, A. Baktir
Pectinase is an enzyme that can hydrolyze substances of pectin. It has several applications in daily life, for example in juice factories, wine factories, pulp and paper factories, coffee and tea factories that use fermentation. Pectinase consists of pectate lyase, pectin lyase, and polygalacturonase. The aim of this research is to partially purify pectinase protein by ammonium sulfate precipitation and dialysis. A preliminary test of pectinase activity used halo zone analysis. The partial purification was needed to optimize ammonium sulfate and get the optimal condition in 40–90% of the ammonium sulfate composition. Pectinase activity was measured using DNS method, and the concentration of protein was measured using Bradford method. The result of this partial purification of Anoxybacillus flavithermus TP-01 was 70%. Decreasing od pectinase activity was resulted in this research due to the loss of cofactor. Based on the result, it can be concluded that the pectinase from Anoxybacillus flavithermus TP-01 was polygalacturonase.
{"title":"Partial Purification of Thermophilic Pectinase from Local Isolate Anoxybacillus flavithermus TP-01","authors":"Asni Putri, Galih Ayhusta Laras, Ismi Aulia Syafira, A. Baktir","doi":"10.20473/jbiome.v1i1.35861","DOIUrl":"https://doi.org/10.20473/jbiome.v1i1.35861","url":null,"abstract":"Pectinase is an enzyme that can hydrolyze substances of pectin. It has several applications in daily life, for example in juice factories, wine factories, pulp and paper factories, coffee and tea factories that use fermentation. Pectinase consists of pectate lyase, pectin lyase, and polygalacturonase. The aim of this research is to partially purify pectinase protein by ammonium sulfate precipitation and dialysis. A preliminary test of pectinase activity used halo zone analysis. The partial purification was needed to optimize ammonium sulfate and get the optimal condition in 40–90% of the ammonium sulfate composition. Pectinase activity was measured using DNS method, and the concentration of protein was measured using Bradford method. The result of this partial purification of Anoxybacillus flavithermus TP-01 was 70%. Decreasing od pectinase activity was resulted in this research due to the loss of cofactor. Based on the result, it can be concluded that the pectinase from Anoxybacillus flavithermus TP-01 was polygalacturonase.","PeriodicalId":371756,"journal":{"name":"Journal of Bio-Molecule Research and Engineering","volume":"66 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2022-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"126297444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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