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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 IS HELIANTI; MARIA ULFAH; NIKNIK NURHAYATI; LLINA MULYAWATI
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.
Medium Optimization for Penicillin Acylase (PAc) Production by Recombinant B. megaterium MS941 Containing pac Gene from B. thuringiensis BGSC BD1 Using Response Surface Methodology FENTRI PARAMITHA PUTRI; ASTUTIATI NURHASANAH; NIKNIK NURHAYATI; IS HELIANTI; KHASWAR SYAMSU
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.
Application of Response Surface Method in Optimization of Medium Composition for Xylanase Production by Bacillus halodurans CM1 in Submerged Fermentation SARA GUSTIA WIBOWO; IS HELIANTI; ANI SURYANI; BUDIASIH WAHYUNTARI
Microbiology Indonesia Vol. 10 No. 3 (2016): September 2016
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.10.3.5

Abstract

A two level factorial design was performed to optimized xylanase production by alkalothermophilic Bacillus halodurans CM1 using response surface method. The variables involved in this experiment were carbon (X), 1 nitrogen source (X) concentration, and pH (XCorn cob and fish powder were use as carbon and nitrogen source 2 3 respectively. Statistical analysis of the experimental results in the range studied, only carbon source gave significant effect on xylanase production.  A second-order model was proposed to represent the enzyme activity as a function of xylan concentration (X) and pH (X).  The optimum corn cobs concentration was 4.37% (w/v), 1 3 fish powder P concentration was 1.75% (w/v) and pH 9. These conditions were tested and validated experimentally since the maximum growth rate achieved with these parameters, and the highest xylanase activity.
The utilization of auto-inducible Plyb promoter and media optimation for cell density-dependent expression of recombinant xylanase in Bacillus subtilis DB104 Haniyya Haniyya; Dini Achnafani; Maria Ulfah; Niknik Nurhayati; Is Helianti
Microbiology Indonesia Vol. 14 No. 1 (2020): March 2020
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.14.1.2

Abstract

Strong promoters are one of the fundamental aspects to increase the level of gene expression, and one of approach to improve the recombinant enzyme productivity so that the efficiency of production cost for enzyme production in industrial scale can be reached. Here we assessed the application of a cell density-dependent promoter and media optimation to promote cell growth and protein expression of Bacillus subtilis without excess usage of inducers. An auto-inducible Pylb promoter that is potential to provide inducer-free enzyme production was cloned and introduced into xylanase recombinant system in B. subtilis DB104 by PCR cloning and protoplast transformation. A 200 bp target gene was successfully inserted in between xynCM1 ORF -coding for B. halodurans CM1 xylanase- and its native promoter sequence at the upstream region. The disruption of the native promoter was intended to replace the native promoter with Pylb. Recombinant xylanase gene under Pylb was successfully expressed in B. subtilis DB104 and the enzyme was produced at stationary phase. Different media with various concentrations of glucose and nitrogen were used to optimize recombinant xylanase expression. It achieved a higher level of xylanase expression compared to wild-type and recombinant xylanase with native promoter B. subtilis in media containing a 2-fold recipe of LB media thus leads to increase cell density and xylanase expression (81.461 U mL-1).
Production and Characterization of Thermoalkaliphilic Xylanase from Bacillus halodurans CM1 on Degumming Process of Ramie (Boehmeria nivea L.Gaud)Fiber as Textile Raw Material DEWI NANDYAWATI; DEA INDRIANI ASTUTI; NIKNIK NURHAYATI; ASEP RISWOKO; IS HELIANTI
Microbiology Indonesia Vol. 15 No. 3 (2021): September 2021
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.15.3.3

Abstract

Ramie fiber is a potential raw material to substitute imported raw materials such as cotton. Due to its higher hemicellulose content, ramie fiber required hydrolysis in a process called degumming. Enzymatic degumming is environmentally friendly compared to traditional process which using chemicals. Alkalithermophilic xylanase have high ability in hemicellulose hydrolysis. The production of xylanase was conducted by submerged fermentation of Bacillus halodurans CM1 in 20L bioreactor using Mamo and corncob medium with optimum conditions at 50°C, pH 9, 150 RPM and 1 vvm. The optimum specific activity of xylanase measured by Bailey method at 70°C and pH 9 is 475.41 U/mg. Xylanase was stable at 50°C, pH 9 and relatively stable to K+, Na2+, Co2+ and Ca2+ metal ions and Triton-X, Saba dan Tween-80 surfactants. Degumming process was carried out by immersing ramie fibers in formulated degumming solution with vlot 1:20 at 50°C, 150 RPM and 180 minutes. The enzymatic degumming process may substitute or reduce the use of chemicals due to its significant effect on ramie fiber quality. Enzymatic and chemical degumming process reduce the weight of Ramie Fiber to 7.23 %, and 7.72 %, slightly higher than enzymatic degumming 7.15%. Enzymatic degumming maintains tensile strength at 27.51 %. Whiteness index enhanced to 2.99% enzymatically and 3.49% chemically. Keywords: Bacillus halodurans CM1, enzymatic degumming, ramie fiber, textile industry, thermoalkaliphilic xylanase
Gene Cloning of Xylanase Glycoside Hydrolase Family 11 from Bacillus halodurans CM1 in Escherichia coli DH5α Muhamad Taufiqul Naufal; Agustin Krisna Wardani; IS HELIANTI
Microbiology Indonesia Vol. 13 No. 4 (2019): December 2019
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.13.4.3

Abstract

Xylanase is an enzyme that can break down xylan into xylose and xylooligosaccharide that is widely used in industry. Seeing the many applications of this enzyme, researchers conducted many studies on how to increase the productivity and effectiveness of the xylanase enzyme. One of the method that can be used to increase the xylanase enzyme production process is by using recombinant DNA technology such as cloning. Bacillus halodurans CM1 is a local alkalothermophilic bacterium that potential producer for xylanase and other industrial enzymes. This research was conducted to clone the GH11 xylanase coding gene from B. halodurans CM1 using pJET 1.2 / blunt plasmid as vector into Escherichia coli DH5α as cell host and  determine the nucleotide base sequence of the GH11 xylanase coding gene from B. halodurans CM1. The results showed the GH11 xylanase gene from B. halodurans CM1 was successfully cloned in  E. coli DH5α and based on the results of BLAST nucleotides had 99% similarities with that of endo-1,4-beta -xylanhydrolase (xyn11A) from B. halodurans C-125. Key words: Bacillus halodurans CM1, cloning, xylanase glycoside hydrolase family 11
Direct Cloning of a Xylanase Gene from Pawan-Riau Hot Spring IS HELIANTI
HAYATI Journal of Biosciences Vol. 14 No. 2 (2007): June 2007
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (137.564 KB) | DOI: 10.4308/hjb.14.2.54

Abstract

A functional gene containing an Open Reading Frame (ORF) encoding a ?-1, 4-endoxylanase glycosyl hydrolase family 11 was cloned directly using metagenomic PCR-cloning method from Pawan Hot Spring sample in Riau. The gene consisted of 642 nucleotides, encoded for 213 amino acids. The amino acid sequence analysis using BLAST showed that the gene has high homology (93%) with xylanase gene from Bacillus subtilis. The gene showed its function when it was subcloned into an expression vector and overexpressed in E. coli. The crude extract of the recombinant enzyme had activity for 170 U/ml at 50 oC. The result of this work showed that metagenomic approach was a powerful short cut method to obtain recombinant biocatalyst that was useful for industrial application. Key words: ?-1, 4-endoxylanase, metagenomic DNA, Pawan-Riau hot-spring
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 MOCHAMAD NURCHOLIS; NIKNIK NURHAYATI; IS HELIANTI; MARIA ULFAH; BUDIASIH WAHYUNTARI; AGUSTIN KRISNA WARDANI
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.
Recent Developments in the Bioconversion of Lignocelluloses into Ethanol . KOESNANDAR; IS HELIANTI; NIKNIK NURHAYATI
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.
Cloning and Gene Expression of AnsZ Encoding L-Asparaginase Enzyme from Local Bacillus sp. RIMA AZARA; IS HELIANTI; JONI KUSNADI; YUNIANTA YUNIANTA
Microbiology Indonesia Vol. 8 No. 2 (2014): June 2014
Publisher : Indonesian Society for microbiology

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5454/mi.8.2.1

Abstract

L-asparaginase is an enzyme that catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. In medical aspect, L-asparaginase especially those came from E. coli and Erwinia chrysanthemi used as chemotherapy agent of acute lymphoblastic leukemia (ALL). However, new potential organisms possessing L-asparaginase production capacity with a similar therapeutic effect are still required . In Bacillus subtilis strain 168, there are two kinds of L-asparaginase gene, AnsA and AnsZ. The study of the later L-asparaginase (AnsZ) has not been conducted intensively. The aim of this study is, first, to isolate this gene of L-asparaginase (AnsZ) from local Bacillus sp. and then to express this gene in Escherichia coli. Using PCR-cloning method, an open reading frame (ORF) containing 1128 bp was obtained. The ORF has 99% homology with sequence of L-asparaginase from Bacillus subtilis Bsn5. The gene then was subcloned into pET 21d (+) with his6-tag in the C-terminal of the gene product and expressed in E.coli BL21. L-asparaginase activity analyses showed that recombinant E. coli containing recombinant plasmid with open reading frame (ORF) L-asparaginase (AnsZ) from Bacillus subtilis had higher activity than that is not containing ORF L-asparaginase (AnsZ). Purification with HisPur TM Ni-NTA Purification Kit increased the specific activity of L-asparaginase (AnsZ) enzyme to 29 fold.