NIKNIK NURHAYATI
Center for Bioindustrial Technology, Laboratorium of Bioindustrial Technology, LAPTIAB BPPT Puspiptek -Serpong

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Recent Developments in the Bioconversion of Lignocelluloses into Ethanol KOESNANDAR, .; HELIANTI, IS; NURHAYATI, NIKNIK
Microbiology Indonesia Vol 2, No 3 (2008): December 2008
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.2.3.1

Abstract

Ethanol has been commercially produced using sugars derived from sugarcane and corn. Recently, research has been focused on the development of thermotolerant and ethanol-tolerant yeast or bacteria that are able to produce ethanol efficiently, as well as the development of lignocellulosic materials as the carbon sources of fermentation. Utilization of lignocellulosic materials as fermentation substrate is promising since they are available in large amounts, renewable and relatively cheap. A lignocellulose biomass is a complex mixture of carbohydrate polymers. In order to develop an efficient process, there have been many attempts to obtain more efficient ways in the conversion of lignocelluloses to ethanol, including pretreatment, enzymatic hydrolysis of lignocelluloses and direct co-culture fermentation. This paper describes the production process of ethanol from starch-containing material, recent developments on the enzymatic bioconversion of lignocelluloses into sugars and their subsequent fermentation into ethanol and the possible recombination of microbes for the direct conversion of lignocelluloses into ethanol.
Characterization of a New Thermoalkalophilic Xylanase-Producing Bacterial Strain Isolated from Cimanggu Hot Spring,West Java, Indonesia ULFAH, MARIA; HELIANTI, IS; WAHYUNTARI, BUDIASIH; NURHAYATI, NIKNIK
Microbiology Indonesia Vol 5, No 3 (2011): September 2011
Publisher : Indonesian Society for microbiology

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Abstract

Alkalophilic bacteria and their enzymes are important for industrial applications. Therefore, finding out new strains of alkalophilic bacteria from Indonesian microbial diversity is still required. In this study, a thermoalkalophilic bacterium was isolated from sediment of local hot spring, Cimanggu,West Java. The temperature of the spot was 60 °C and the pH was 8. The bacterium could live at pH 11 and temperature 55 °C, and produced xylanase that have optimal activity at alkaline pH 9 and high temperature 70 °C. Based on the analyses of 16S rRNA sequence similarity and biochemical characteristics, this strain was clustered into the same group of Bacillus halodurans species with 99% identity to Bacillus halodurans C-125. The isolate also showed other enzyme activities such as amylase, protease, and gelatinase, promising its potential use as an industrial enzymes producer.
Cloning of α-L-arabinofuranosidase Genes and Its Expression in Escherichia coli: A Comparative Study of Recombinant Arabinofuranosidase Originatingin Bacillus subtilis DB104 and Newly Isolated Bacillus licheniformis CW1 NURCHOLIS, MOCHAMAD; NURHAYATI, NIKNIK; HELIANTI, IS; ULFAH, MARIA; WAHYUNTARI, BUDIASIH; WARDANI, AGUSTIN KRISNA
Microbiology Indonesia Vol 6, No 1 (2012): March 2012
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.6.1.1

Abstract

Arabinofuranosidase (Abfa) is one of the most important enzymes involved in degradation of lignocelullose biomass.  Two genes encoding α-L-Arabinofuranosidase (abfA), each from Bacillus subtilis DB104 (abfAa1) and an indigenous Indonesian B. licheniformis CW1 (abfAb3), were cloned by the PCR approach  and expressed in Escherichia coli. Sequences analysis of abfAa1 and abfAb3 revealed that each consists of 1721 and 1739 base pairs long DNA, respectively. Each clone contains a hypothetical open reading frame of 1503 and 1509 bp that encode an Abfa protein of 500 and 502 amino acids for abfAa1 and abfAb3, respectively. The deduced amino acid sequence of AbfaB3 shares 75% identity to that of AbfaA1. The recombinant enzymes were expressed constitutively in E. coli. Partial characterization of those enzymes revealed that the AbfaA1 and AbfaB3 were optimally active at 50 ºC and 60 ºC at pH 6, respectively. Thermostability studies of the recombinant enzymes with p-nitrophenyl α-L-arabinofuranoside at their optimal conditions showed that up to 50% AbfaA1 activity was lost after 5 h incubation at 50  ºC, whereas the AbfaB3 retained its activity over 75% after 12 h pre-incubation oat 60 ºC. This thermostability study of recombinant AbfaB3 showed for the first time that the arabinofuranosidase from B. licheniformis is a thermostable enzyme. The recombinant enzyme showed a higher optimal reaction temperature (60 ºC) in comparison to the previously reported thermostable arabinofuranosidase. The thermostable AbfaB3 has a potential to be applied to the degradation of lignocellulose biomass synergistically with thermostable xylanases, for instance in the production of xylo-oligosaccharides.
Cloning, Sequencing, and Expression of the Gene Encoding a Family 9 Cellulase from Bacillus licheniformis F11 in Escherichia coli and Bacillus megaterium, and Characterization of the Recombinant Enzymes HELIANTI, IS; ULFAH, MARIA; NURHAYATI, NIKNIK; MULYAWATI, LLINA
Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.8.4.2

Abstract

 A gene encoding cellulase belonging to the glycosyl hydrolase family 9 along with its native promoter was isolated from Bacillus licheniformis F11, cloned in Escherichia coli DH5 α and subcloned by transconjugation to Bacillus megaterium MS941. Functionality of the encoded protein was proven both in heterologous hosts, E. coli and B. megaterium. In the latter, the gene product was found in the extracellular fraction expressing a high specific activity; whereas in E. coli the protein was not secreted into the medium, and rather, showed a lower specific activity. The optimum temperature of the recombinant enzyme expressed in the hosts range from 65-75 ºC; whereas the optimum pH is 6. The recombinant enzyme was stable between 50-60 ºC and in a broad pH range (pH 5 - 9). Addition of Ca2+ and Fe3+ enhanced the enzyme activity, whereas EDTA and Cu2+  had the opposite effect. Lichenin, rather than carboxyl methyl cellulose, is the preferred substrate.
PRODUCTION OF XYLANASE BY RECOMBINANT BACILLUS SUBTILIS DB104 CULTIVATED IN AGROINDUSTRIAL WASTE MEDIUM Helianti, Is; Ulfah, Maria; Nurhayati, Niknik; Suhendar, Dadang; Finalissari, Anita Kusuma; Wardani, Agustin Krisna
HAYATI Journal of Biosciences Vol. 23 No. 3 (2016): July 2016
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1064.942 KB) | DOI: 10.4308/hjb.23.3.125

Abstract

A recombinant Bacillus subtilis DB104 strain harbouring recombinant plasmid pSKE194 containing an Open Reading Frame (ORF) of endoxylanase and its indigenous promoter from the wild-type B. subtilis AQ1 strain was constructed. This recombinant B. subtilisDB104 strain had higher endoxylanase activity than the nonrecombinant B. subtilisDB104 strain in standard media, such as Luria Bertani (LB) and LB with xylan. The agroindustrial wastes corncobs and tofu liquid waste were chosen as cost-effective carbon and nitrogen sources, respectively, to test the economics of xylanase production using the recombinant B. subtilis DB104 at a larger scale. Submerged fermentation using a 4.5 L working volume fermentor with tofu liquid waste and 4% corncobs produced maximum xylanase activity of 1296 ± 1.2 U/mg (601.7 ± 0.6 U/mL) after 48-hour fermentation at 37°C with 150 rpm agitation; this is more than twofold higher than the activity produced in an Erlenmeyer flask. This is the first report of high xylanase activity produced from recombinant B. subtilis using inexpensive medium. During fermentation, the xylanase degrades corncobs into xylooligosaccharides, showing its potential as an enzyme feed additive or in xylooligosaccharide production.
Cloning of Synthetic Lipase Gene from Rhizomucor miehei with Original Signal Peptide in Pichia pastoris CAHYANI, MARTHA EKA; HELIANTI, IS; NURHAYATI, NIKNIK; ABINAWANTO, ABINAWANTO
Microbiology Indonesia Vol 11, No 1 (2017): March 2017
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.11.1.1

Abstract

Lipases (EC 3.1.1.3) are classified as hydrolases that hydrolyze lipids. These enzymes have potential application in biotechnology and industrial process. In previous study we have cloned the synthetic Rhizomucor miehei lipase gene using the vector pUC57 in Escherichia coli DH5α, but only found the very low enzymes activity. This study aimed to clone Rhizomucor miehei synthetic lipase gene into Pichia pastoris expression plasmid for lipase expression with the original signal peptide. A DNA fragment with the original signal peptide had been obtained by PCR, cut by Xho I and Xba I and then ligated into pPICZα A linearized with the same enzymes. The mixture of ligation, then was transformed into Escherichia coli DH5α. Zeocin-resistant transformants were selected and contained plasmid was analyzed by restriction enzymes analyses, PCR, and DNA sequenced. As the result, a Rhizomucor miehei lipase gene (RMlip) with the size of 1132 bp was successfully cloned to pPICZα A. The recombinant plasmid with the correct DNA sequence was transformed into Pichia pastoris X33. Cultivation of recombinant P. pastoris was carried out with the addition of 1.5% methanol every day with appropriate aeration. The recombinant lipase produced by Pichia pastoris X33 containing RMlip oin its chromosomal DNA had optimal temperature and pH 30 C and 9.0, respectively.
Medium Optimization for Penicillin Acylase (PAc) Production by Recombinant B. megaterium MS941 Containing pac Gene from B. thuringiensis BGSC BD1 Using Response Surface Methodology PUTRI, FENTRI PARAMITHA; NURHASANAH, ASTUTIATI; NURHAYATI, NIKNIK; HELIANTI, IS; SYAMSU, KHASWAR
Microbiology Indonesia Vol 9, No 2 (2015): June 2015
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.9.2.3

Abstract

Penicillin G acylase (PAc) hydrolyses of the amide bond of benzylpenicillin (Pen-G) releasing PAA and 6-APA, key intermediate in the production of various semisynthetic penicillins. In this study, we optimised the production medium of PAc by RSM using two variables (xylose as inducer and CaCl2 as divalent cations) to obtain the optimum PAc specific activity from Bacillus megaterium btpacBD1. For this purpose, combinations of five different xylose concentrations (0.13 – 0.87 %) and five different CaCl2 concentrations (0.64 – 4.36 mM) were analysed, in a total of 22 experiments. CCD used for the analysis showed that in shake flask cultivations, xylose and CaCl2 showed significant effects on PAc volumetric activity and the quadratic model was in good agreement with the experimental results (R2= 0.86 (p-value < 0.0001)). The maximum specific activity (130.669 ± 50.241 units mg protein-1) was reached when xylose and CaCl2 concentrations were 0.49% and 2.4 mM, respectively, and medium pH was around 7. Under such conditions, the activity of PAc and protein concentration achieved were 1.318 ± 0.406 units mL-1 and  0.0101 ± 0.01 mg mL-1. The shake flask validation experiments demonstrated that with such medium composition the volumetric activity, protein concentration and specific activity achieved were 1.294 ± 0.171 units mL-1, 0.0102 ± 0.0003 mg mL-1 and 125.91 ± 13.309 units mg-1, respectively. When the optimum medium composition was applied in 10 L bioreactor, the optimum volumetric activity (2.0687 ± 0.0820 units mL-1) and protein concentration (0.0078 ± 0.0008 mg mL-1) were achieved 48 h after the start of the cultivation. However, the optimum PAc specific acivity (1260.52  ± 27.5711 units mg protein-1) was achieved 18 h after the start of the cultivation.