Recently, studies were initiated to investigate the metagenome, which represents the genomes of cultured and uncultured microbes, as a rich source for isolation of many novel genes. The metagenomic approach originated from the molecular analysis of microbial communities, which revealed that the majority of microorganisms in nature were not cultivable by standard culturing techniques. Therefore, most microorganisms in nature have not been characterized. Although numerous methods have been reported for direct DNA isolation and purification from microorganisms in soil, the sample preparation procedures and experimental conditions used in different studies vary widely. Soils are therefore one of the most challenging environmental matrices from which to obtain microbial DNA that will support PCR. The Papaloapan River is the second largest river basin in México. For the climatic conditions of this region, there is great diversity in plants, animals and microorganisms. In the Papaloapan region different fruits are grown, however, the main crops are sugarcane and pineapple. In this work the extraction of DNA from soils of sugarcane cultivation was performed. We used PCR tests to assess the quality of DNA extracted from soil by amplifying the 16S rDNA gene. Changes in both protocols were performed; satisfactory results were obtained as to the quality of DNA and gene amplification. These results will allow continuing the metagenomic studies, such as sequencing, library construction and identification of enzymes cellulase and amylase activity. It is the first time these studies were performed in the Papaloapan region.

Bacillus amyloliquefaciens strain JJC33M is a bacterium that produces α-amylase (EC 3.2.1.1) and was isolated from sugarcane soil. Its estimated genome size is 3.96 Mb, and it harbors 4,048 coding genes (CDSs).

La amilasa (E.C. 3.2.1.1) de Bacillus amyloliquefaciens JJC33M (AmiJ33) fue producida por fermentación sumergida. Se probó el efecto de peptona, extracto de levadura, Ca+2 y glicina en la producción de AmiJ33. El extracto de levadura y Ca+2 tuvieron un efecto positivo sobre la síntesis de AmiJ33. La enzima fue recuperada mediante precipitación a saturación al 60% con (NH4)2SO4. El peso molecular aproximado de la enzima purificada fue de 50 kDa. Así mismo, se evaluó el efecto del pH y la temperatura sobre la actividad enzimática, concluyendo que los valores más altos de actividad se observaron a pH 6.0 y 80°C, respectivamente. En condiciones ligeramente ácidas (pH 4.0 y 5.0), AmiJ33 mantuvo el 72% de su actividad. AmiJ33 fue estable por 3 h a 40°C, y por 30 min a 45 y 50°C, conservando el 88 y 82% de actividad residual. A 60°C, la actividad disminuyó 40%. La actividad de AmiJ33 se incrementó 50.27% con b-mercaptoetanol, no fue inhibida por EDTA y se inhibió totalmente con SDS

AmyJ33, an α-amylase isolated from JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.