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The Acidiphilum multivorum strain AIU 301 pKW301 plasmid was transferred to Escherichia coli JM109 strain to determine the role of the plasmid in arsenic resistance. Analysis of the transformed Escherichia coli strain containing the pKW301 plasmid indicated the role of large plasmid in conferring resistance to arsenic and mercuric chloride. Furthermore, the plasmid contained genes that make host cells resistant to heavy metal salts.

The characterization of two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, purified from Schizosaccharomyces pombe IFO 0354, shows that they are immunologically different from each other. DHAK II is more important than DHAK I for the dissimilation of glycerol via dihydroxyacetone. When the enzyme activity is measured in a mixed buffer, DHAK I shows optimum activity at pH 6.0 and DHAK II at pH 7.0. The optimum temperature for the activity of DHAK I is 50 degree celsius (C)and that for the activity for DHAK II is 60 degree C.

The mutant strain Ruminococcus albus A3 was introduced into the rumen of a goat and its survival was tested to determine how long antibiotic-resistant cellulose using bacteria can survive in the rumen. The number of bacteria decreased sharply after one day possibly because of the presence of protozoan predators and a low growth rate. In the rumen of the goat, the bacteria survived for 14 days, while in a simulated rumen, the bacteria survived for only 96 hours and the number of surviving cells was less.

Research has been conducted on Clostridium thermocellum CelJ which contains a family 30 carbohydrate-binding module. The functions of this module and of the catalytic module have been investigated via characterizarion of constructed CelJ truncated derivatives, and the details are reported.

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10–30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements. [ABSTRACT FROM AUTHOR]

Laminin_G_3 modules can exist together with family‐43 catalytic modules of glycoside hydrolase (GH43), but their functions are unknown. Here, a laminin_G_3 module and a GH43 module derived from a Ruminiclostridium josui modular arabinofuranosidase Abf43A‐Abf43B‐Abf43C were produced individually as RjLG3 and RjGH43_22, respectively, or combined as RjGH43‐1 to gain insights into their activities. Isothermal calorimetry analysis showed that RjLG3 has high affinity toward 32‐α‐l‐arabinofuranosyl‐(1,5)‐α‐l‐arabinotriose but not for α‐1,5‐linked arabinooligosaccharides, which suggests that RjLG3 interacts specifically with a branched arabinofuranosyl residue of an arabinooligosaccharide but not an arabinofuranosyl residue at the end of α‐1,5‐linked arabinooligosaccharides. RjGH43‐1 (with CBM) shows higher activity toward sugar beet arabinan than RjGH43_22 (without CBM), which suggests that the LG3 module in RjGH43‐1 plays an important role in substrate hydrolysis as a carbohydrate‐binding module. [ABSTRACT FROM AUTHOR]

Although Ruminiclostridium josui (formerly Clostridium josui), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host‐vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui. Here, we partially purified restriction endonuclease RjoI from R. josui cell extract using column chromatography. Further characterization showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5′‐GmetATC‐3′, where the A nucleotide is Dam‐methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 (dam−/dcm−) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 103 transformants/μg of DNA was obtained using a square‐wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin‐encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui. [ABSTRACT FROM AUTHOR]

Bacillus safensis M3 was newly isolated from freshwater swamp forest soil in western Thailand. The endoglucanase gene of B. safensis M3, cel9A, had an open reading frame of 1,848 bp encoding a 616 amino acid protein. Initial expression in Escherichia coli yielded a low amount of soluble protein in the cytosolic and secreted fractions. Cel9A was successfully expressed by recombinant B. subtilis with a 4- fold greater total enzyme activity than from recombinant E. coli. By SDS-PAGE analysis, the molecular weight of Cel9A was estimated to be 70 kDa. The optimal temperature of Cel9A was 55 °C and the optimal pH was 5 - 8. Cel9A had the highest activity in the pH range from 5 - 8 and the highest stability in pH range 4 to 10, which is useful for industrial applications. Notably, Cel9A was able to hydrolyze both mixed linkage glucan (lichenan) and hemicellulose (konjac glucomannan and oat spelt xylan) better than carboxymethylcellulose. Cel9A also showed a tolerance to metal ions and surfactants. In addition, recombinant B. subtilis with endoglucanase activity has potential for biotechnological applications and benefits in the optimization of large scale enzyme production with minimal medium using agriculture wastes and other inexpensive feedstock materials. [ABSTRACT FROM AUTHOR]

The newly isolated Paenibacillus sp. M33 from freshwater swamp forest soil in Thailand demonstrated its potential as a cellulose degrader. One of its endoglucanase genes from Paenibacillus sp., celP, was cloned to study the molecular characteristics of its gene product. The celP gene was recognized firstly by degenerate primer designed from Paenibacillus endoglucanase gene, and subsequently identified flanking region by inverse PCR technique. The celP gene consists of an open reading frame of 1707 bp encoding for 569 amino acids including 33-amino acids signal sequence. CelP is a member of glycoside hydrolase family 5 appended with a family 46 carbohydrate-binding module. CelP from recombinant Escherichia coli was purified by affinity chromatography. SDS-PAGE analysis of purified CelP showed a protein band at about 60 kDa. The purified enzyme gave a specific CMCase activity of 0.03 μmol min−1 mg−1. It had higher activities on lichenan (0.19 μmol min−1 mg−1) and barley β-glucan (0.14 μmol min−1 mg−1). Maximum activity on lichenan was obtained at 50 °C, pH 5.0. CelP was stable over a pH range of 3.0-10.0 and retained 80% activity when incubated at 50 °C for 1 h. The properties of its CelP endoglucanase, especially substrate specificity, will make it useful in various biotechnological applications including biomass hydrolysis. [ABSTRACT FROM AUTHOR]

Carbohydrate binding modules (CBMs) are commonly known to have the potential to increase binding of carbohydrate-active enzymes to target polysaccharides, thus the catalytic module can easily contact the substrate via CBM. Laccase is one of the most important enzymes used for delignification step in paper industry. This enzyme improves the product's quality, industrial process, and environmentally sound technologies. In this study, we have constructed the fusion gene encoding a novel CBM36 from β-Xylosidase B (GbtXyl43B) of Geobacillus thermoleovorans IT-08 and laccase (LCC2) of Pleurotus salmoneostramineus using overlapping polymerase chain reaction (PCR), and inserted into expression vectors pET-32a and pYHM1 for Escherichia coli BL21 codon plus and Saccharomyces cerevisiae BJ1824, respectively. The fusion protein, Cbm36-Lcc2, was successfully expressed both in E. coli and S. ceerevisiae and optimum pH and temperature were determined to be 5.0 and 60 °C, and 6.0 and 60 °C, respectively, with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate; ABTS) as the substrate. The thermostability and pH stability indicated that Cbm36-Lcc2 expressed in S. cerevisiae BJ1824 was more stable than in E.coli BL21 codon plus. The laccase activity for Cbm36-Lcc2 was two to three times higher than that for Lcc2 itself. This study developed the new insight that fusion of novel Cbm36 into Lcc2 could improve the thermostability of Lcc2 in its chimeric laccase form of Cbm36-Lcc2 compared to Lcc2 itself. • Laccase is one of enzymatically innovations for delignification processes that are more efficient and eco-friendly. • The novel CBM36 gene was successfully fused to LCC2 using an overlapping PCR, and could be expressed in heterologous host. • New insight displayed that the chimeric laccase could improve its thermostability and activity than that for Lcc2 itself. [ABSTRACT FROM AUTHOR]