Amare Gessesse

Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather

Two alkaline protease producing alkaliphilic bacterial strains, designated as AL-20 and AL-89, were isolated from a naturally occurring alkaline habitat. The two strains were identified as Nesternkonia sp. and Bacillus pseudofirmus, respectively. Both strains grew and produced alkaline protease using feather as the sole source of carbon and nitrogen. Addition of 0.5% glucose to the feather medium increased protease production by B. pseudofirmus AL-89 and suppressed enzyme production by Nesternkonia sp. AL-20. The enzymes from both organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight, determined using SDS–PAGE, was 23 kDa for protease AL-20 and 24 kDa for protease AL-89. Protease AL-20 was active in a broad pH range displaying over 90% of its maximum activity between pH 7.5 and 11.5 with a peak at pH 10. The enzyme is unique in that unlike all other microbial serine proteases known so far, it did not require Ca2+ for activity and thermal stability. Its optimum temperature for activity was at 70 °C and was stable after 1 h incubation at 65 °C both in the presence and absence of Ca2+. These properties make protease AL-20 an ideal candidate for detergent application. Protease AL-89 on the other hand require Ca2+ for activity and stability at temperature values above 50 °C. Its optimum activity was at 60 and 70 °C in the absence and presence of Ca2+, respectively. It displayed a pH optimum of 11 and retained about 70% or more of its original activity between pH 6.5 and 11. B. pseudofirmus AL-89, and the protease it produce offers an interesting potential for the enzymatic and/or microbiological hydrolysis of feather to be used as animal feed supplement.

High-level xylanase production by an alkaliphilic Bacillus sp. by using solid-state fermentation

Bacillus sp. AR-009 produced up to 720 U/g dry bacterial bran xylanase activity when grown by using solid-state fermentation with wheat bran serving as a substrate. Xylanase production was highest at a wheat bran-to-moisture ratio of from 1:0.5 to 1:1.5 and an Na2CO3 concentration of 10% (w/w). Strong repression of xylanase production was observed in the presence of 5% (w/w) xylose and lactose, whereas sucrose and glucose at the same concentration slightly affected enzyme production. The effect of glucose was concentration-dependent, inducing less than 10% of the maximum xylanase production at a concentration of 15% (w/w). No significant effect was observed on xylanase production upon addition of peptone and tryptone, whereas yeast extract slightly stimulated enzyme production. The ability of the organism to produce high-titer xylanase activity at alkaline pH and lower wheat bran-to-moisture ratio could have a potential advantage in minimizing the risk of contamination. In addition, because the enzyme could be extracted by using a minimum volume of liquid, the cost of downstream processing during product upgrading and the cost of waste treatment steps can be greatly reduced. The use of solid-state fermentation for the production of xylanase by Bacillus sp. AR-009 could, therefore, lead to substantial reduction in the overall cost of enzyme production.

Lipase and protease extraction from activated sludge

In the process of wastewater treatment hydrolysis of polymeric substances is the first and rate-limiting step. A closer study of the enzymes catalysing these reactions is essential for a better understanding of the microbial activity in the wastewater treatment process. Therefore, development of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination for the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase activity and negligible protease activity was extracted in the presence of buffer alone, indicating that enzyme extraction was not due to shear force alone. The highest lipase activity was extracted using 0.1% Triton X-100 above which the activity was gradually decreasing. For proteases, the highest activity was obtained in the presence of 0.5% Triton X-100 and no decrease in activity was observed. Differences observed in the extraction efficiency of the two enzymes indicate the need for optimisation of the extraction process for the different enzymes or the extracellular polymeric substances from activated sludge.

Production of alkaline protease by an alkaliphilic bacteria isolated from an alkaline soda lake

A new alkaliphilic strain of Microbacterium producing an alkaline protease was isolated from an alkaline soda lake in Ethiopia. High level of protease activity was produced in the presence of glucose and sucrose as carbon sources. The optimum temperature and pH for activity were 65°C and 9.5-11.5 respectively. Above 50°C, Ca2+ was required for enzyme activity and stability. At 55 and 60°C it retained 100 and 85% of its original activity respectively after 1 h incubation. The enzyme was stable over the pH range of 5-12.

The use of nug meal as a low-cost substrate for the production of alkaline protease by the alkaliphilic Bacillus sp. AR-009 and some properties of the enzyme

Bacillus sp AR-009 produced appreciable extracellular alkaline protease when grown using nug meal (a byproduct of oil extraction from Guizotia abyssinica seeds) as the sole nitrogen source. The enzyme had an optimum pH of 9.5–11.5 and was stable in the pH range of 5–12. Its optimum temperature was 55°C in the absence of calcium and 65°C in the presence of calcium. At 50°C and above calcium was required for thermal stability. The enzyme was completely inhibited by PMSF suggesting that it is a serine protease.

Purification and characterization of two raw-starch-digesting thermostable α-amylases from a thermophilic Bacillus.

Abstract Two α-amylases, designated as AmyI and AmyII, were purified to homogeneity from the cell-free culture supernatant of the thermophilic Bacillus sp. strain WN11. The molecular weights of AmyI and AmyII were estimated to be 76 and 53, respectively. The two amylases completely adsorb to potato and corn starch granules. At an enzyme dose of 1 U/mg raw starch and an incubation temperature of 60°C, the percent hydrolysis of raw potato and corn starch, respectively, was 77 and 44% for AmyI and 82 and 37% for AmyII. The optimum temperature for the activity of both enzymes was at 75–80°C, and ≈50% of the original activity was retained after 4 h of incubation at 80°C. The two enzymes were optimally active at pH 5.5 and stable in the pH range of 5.5–9.0. Activity was inhibited in the presence of Hg2+, Cu2+, and Fe3+, but no inhibition was observed in the presence of Zn2+.

Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkalinesoda lake

An alkaline xylanase‐producing alkaliphilic Bacillus sp. AR‐009 was isolated from analkaline soda lake in Ethiopia. The enzyme was optimally active at pH 9 and was stable over abroad pH range. The optimum temperature for xylanase activity, assayed at pH 9, was60°–65°C. Measured at pH 8 and 9, the enzyme had good stability at 55° and60°C. At both pH values, over 80% of its original activity was retained after heating for2·5 h at 55°C. At 60°C, the enzyme maintained 63% of its original activity after2·5 h incubation while at pH 9 it retained 54% of its original activity after 1 h heating. Theseproperties qualify the enzyme to be novel and potentially important for application in someindustrial processes.

A highly thermostable amylase from a newly isolated thermophilic Bacillus sp. WN11

A thermostable amylase‐producing Bacillus sp. WN11 was isolated from Wondo Genet hot spring in Ethiopia. The enzyme had a temperature optimum of 75–80 °C. Over 80% of its peak activity was in the pH range of 5–8, with an optimum at 5·5. Thermal stability of the enzyme at 105 °C was higher with the addition of starch. The stabilizing effect of starch was concentration‐dependent, showing better stability with increasing concentration of starch. At liquefying temperature (105 °C), addition of Ca2+ did not result in further improvement of the stabilizing effect of starch. This indicates that in the presence of starch, WN11 amylase does not require Ca2+ as a stabilizer at liquefying temperatures as high as 105 °C.

Surprising Prokaryotic and Eukaryotic Diversity, Community Structure and Biogeography of Ethiopian Soda Lakes

Soda lakes are intriguing ecosystems harboring extremely productive microbial communities in spite of their extreme environmental conditions. This makes them valuable model systems for studying the connection between community structure and abiotic parameters such as pH and salinity. For the first time, we apply high-throughput sequencing to accurately estimate phylogenetic richness and composition in five soda lakes, located in the Ethiopian Rift Valley. The lakes were selected for their contrasting pH, salinities and stratification and several depths or spatial positions were covered in each lake. DNA was extracted and analyzed from all lakes at various depths and RNA extracted from two of the lakes, analyzed using both amplicon- and shotgun sequencing. We reveal a surprisingly high biodiversity in all of the studied lakes, similar to that of freshwater lakes. Interestingly, diversity appeared uncorrelated or positively correlated to pH and salinity, with the most “extreme” lakes showing the highest richness. Together, pH, dissolved oxygen, sodium- and potassium concentration explained approximately 30% of the compositional variation between samples. A diversity of prokaryotic and eukaryotic taxa could be identified, including several putatively involved in carbon-, sulfur- or nitrogen cycling. Key processes like methane oxidation, ammonia oxidation and ‘nitrifier denitrification’ were also confirmed by mRNA transcript analyses.

Synthesis of sucrose laurate using a new alkaline protease

Abstract Sucrose laurate esters were synthesized from sucrose and vinyl laurate in organic solvents using an alkaline protease from a new alkalophilic strain, Bacillus pseudofirmus AL-89. Maximum synthetic activity was observed in the presence of 7.5% v/v water and in the pH range of 7–10. With protease AL-89 esterification occurred predominantly at the 2- O -position while subtilisin A-catalyzed monoester formation predominantly at the 1′- O position. In the absence of enzyme, buffer salts catalyzed non-specific reactions, resulting in the formation of a number of esters. Non-specific catalysis was also observed upon inhibition of the enzyme using a serine protease inhibitor or upon deactivation of the enzyme at pH above 10.